Method for robust control of gene expression

ABSTRACT

Disclosed herein include methods, compositions, and kits suitable for robust and tunable control of payload gene expression. Some embodiments provide rationally designed circuits, including miRNA-level and/or protein-level incoherent feed-forward loop circuits, that maintain the expression of a payload at an efficacious level. The circuit can comprise a promoter operably linked to a polynucleotide encoding a fusion protein comprising a payload protein, a protease, and one or more self-cleaving peptide sequences. The payload protein can comprise a degron and a cut site the protease is capable of cutting to expose the degron. The circuit can comprise a promoter operably linked to a polynucleotide comprising a payload gene, a silencer effector cassette, and one or more silencer effector binding sequences.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Ser. No. 62/939,079, filed Nov. 22, 2019,the content of this related application is incorporated herein byreference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under Grant No.HR011-17-2-0008 awarded by DARPA. The government has certain rights inthe invention.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSequence_Listing_30KJ_302423_US, created Nov. 21, 2020, which is 4.0kilobytes in size. The information in the electronic format of theSequence Listing is incorporated herein by reference in its entirety.

BACKGROUND Field

The present disclosure relates generally to the field of polynucleotidedelivery and expression.

Description of the Related Art

Although recent advances have been made in gene manipulation methods andmore efficient delivery methods have been introduced, therapeuticallyeffective gene therapy requires that the payload (e.g., a replacementgene) be expressed in a controlled manner in the target cell(s). Forexample, precise control of MeCP2 expression is required for RettSyndrome gene therapy. Since CRE-induced expression of MeCP2 was shownto reverse the symptoms of Rett syndrome in a mouse model, severalattempts have been made at treating the disease in mice withAdeno-associated virus (AAV)-delivered MeCP2. However, the level ofectopic MeCP2 expression is difficult to control due to various factors,for example variation in gene delivery due to tropism or accessibility;stochasticity in transcription, translation, or other aspects ofexpression; and heterogeneity in the levels of cellular components thataffect protein expression. In Rett Syndrome gene therapy, thisvariability has manifested as toxically high expression of MeCP2 in theliver, while insufficient MeCP2 is expressed in the brain, so thatneither an increase or decrease in the viral dosage of a standard genetherapy vector would be therapeutically effective. However, no genetherapy has been put forth that integrates a rationally designed circuitto control the expression of its payload. There is a need forcompositions, methods, and systems for robust and tunable control ofpayload gene expression.

SUMMARY

Disclosed herein include nucleic acids capable of enabling robust andtunable control of gene expression. In some embodiments, the nucleicacid comprises: a promoter operably linked to a polynucleotide encodinga fusion protein comprising a payload protein, a protease, and one ormore self-cleaving peptide sequences, wherein the payload proteincomprises a degron and a cut site the protease is capable of cutting toexpose the degron, and wherein the degron of the payload protein beingexposed changes the payload protein to a payload protein destabilizedstate. In some embodiments, the promoter is capable of inducing thetranscription of the polynucleotide to generate a payload transcript.

In some embodiments, the degron comprises an N-degron. In someembodiments, the self-cleaving peptide sequence comprises porcineteschovirus-1 2A peptide (P2A), Thosea asigna virus 2A peptide (T2A),equine rhinitis A virus 2A peptide (E2A), foot-and-mouth disease virus2A peptide (F2A), or any combination thereof. In some embodiments, theprotease comprises tobacco etch virus (TEV) protease, tobacco veinmottling virus (TVMV) protease, hepatitis C virus protease (HCVP),derivatives thereof, or any combination thereof. In some embodiments,the protease comprises TEVP, and wherein the cut site comprises theamino acid sequence of ETVFFQ (SEQ ID NO: 1), ENAYFQ (SEQ ID NO: 2),ENLFFQ (SEQ ID NO: 3), ENLYFQ (SEQ ID NO: 4), ENLYFQY (SEQ ID NO: 5),ENLYFQF (SEQ ID NO: 6), ENLYFQQ (SEQ ID NO: 7), or ENLFFQY (SEQ ID NO:8).

Disclosed herein include nucleic acids. In some embodiments, the nucleicacid comprises: a promoter operably linked to a polynucleotidecomprising a payload gene and a silencer effector cassette, wherein thepayload gene 3′UTR comprises one or more silencer effector bindingsequences. In some embodiments, the promoter is capable of inducing thetranscription of the polynucleotide to generate a payload transcript. Insome embodiments, the payload gene encodes a payload protein. In someembodiments, payload gene encodes a siRNA, a shRNA, an antisense RNAoligonucleotide, an antisense miRNA, a trans-splicing RNA, a guide RNA,single-guide RNA, crRNA, a tracrRNA, a trans-splicing RNA, a pre-mRNA, amRNA, or any combination thereof. In some embodiments, the silencereffector cassette comprises a miRNA cassette. The polynucleotide cancomprise: a transcript stabilization element (e.g., hepatitispost-translational regulatory element (WPRE), bovine growth hormonepolyadenylation (bGH-polyA) signal sequence, human growth hormonepolyadenylation (hGH-polyA) signal sequence, or any combinationthereof).

In some embodiments, an intron is located in the payload gene 3′UTR,payload gene 5′UTR, or between payload gene exons, and wherein theintron comprises the silencer effector cassette. In some embodiments,the intron comprises: (i) an intronic insert encoding a silencereffector, (ii) a donor splice site, (iii) an acceptor splice site, (iv)a branch point domain; and (v) a polypyrimidine tract. In someembodiments, said silencer effector is capable of being released fromsaid intron by an intron excision mechanism selected from the groupcomprising cellular RNA splicing and/or processing machinery,nonsense-mediated decay (NMD) processing, or any combination thereof.

In some embodiments, silencer effector comprises a microRNA (miRNA), aprecursor microRNA (pre-miRNA), a small interfering RNA (siRNA), ashort-hairpin RNA (shRNA), precursors thereof, derivatives thereof, or acombination thereof. In some embodiments, the promoter is capable ofinducing the transcription of the polynucleotide to generate a payloadtranscript. In some embodiments, said silencer effector is capable ofbinding the one or more silencer effector binding sequences, therebyreducing the stability of the payload transcript and/or reducing thetranslation of the payload transcript. In some embodiments, the one ormore silencer effector binding sequences comprise miRNA binding sites.In some embodiments, the polynucleotide comprises about 1 silencereffector binding sequence to about 10 silencer binding sequences. Insome embodiments, the one or more silencer effector binding sequencesare about 8 nucleotides to about 22 nucleotides in length. In someembodiments, the silencer effector comprises a region of complementaritythat is complementary with at least 5 consecutive nucleotides of the oneor more silencer effector binding sequences. In some embodiments, thesilencer effector comprises at least about 50% complementarity to theone or more silencer effector binding sequences.

In some embodiments, one or more cells comprise an endogenous version ofa gene encoding the payload protein, and wherein the silencer effectorcomprises at least about 50% complementarity to one or more endogenoussilencer effector binding sequences within the 3′UTR of the endogenousversion. In some embodiments, one or more cells comprise an endogenousversion of a gene encoding the payload protein comprising one or moresecondary silencer binding sequences in the 3′UTR, wherein the nucleicacid comprises a secondary silencer effector cassette encoding asecondary silencer effector that is capable of binding the one or moresecondary silencer binding sequences.

In some embodiments, the promoter comprises a ubiquitous promoter. Insome embodiments, the ubiquitous promoter is selected from the groupcomprising a cytomegalovirus (CMV) immediate early promoter, a CMVpromoter, a viral simian virus 40 (SV40) (e.g., early or late), aMoloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus(RSV) LTR, an RSV promoter, a herpes simplex virus (HSV) (thymidinekinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, anelongation factor 1-alpha (EF1a) promoter, early growth response 1(EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphatedehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1(EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDabeta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin(β-KIN), the human ROSA 26 locus, a Ubiquitin C promoter (UBC), aphosphoglycerate kinase-1 (PGK) promoter, 3-phosphoglycerate kinasepromoter, a cytomegalovirus enhancer, human β-actin (HBA) promoter,chicken β-actin (CBA) promoter, a CAG promoter, a CBH promoter, or anycombination thereof. In some embodiments, the promoter is an induciblepromoter (e.g., a tetracycline responsive promoter, a TRE promoter, aTre3G promoter, an ecdysone responsive promoter, a cumate responsivepromoter, a glucocorticoid responsive promoter, and estrogen responsivepromoter, a PPAR-γ promoter, and/or an RU-486 responsive promoter).

In some embodiments, the promoter comprises a tissue-specific promoterand/or a lineage-specific promoter. In some embodiments, the tissuespecific promoter is a liver-specific thyroxin binding globulin (TBG)promoter, an insulin promoter, a glucagon promoter, a somatostatinpromoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn)promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES)promoter, a α-myosin heavy chain (a-MIC) promoter, or a cardiac TroponinT (cTnT) promoter. In some embodiments, the tissue specific promoter isa neuron-specific promoter (e.g., the neuron-specific promoter comprisesa synapsin-1 (Syn) promoter, a CaMKIIa promoter, acalcium/calmodulin-dependent protein kinase II a promoter, a tubulinalpha I promoter, a neuron-specific enolase promoter, a platelet-derivedgrowth factor beta chain promoter, TRPV1 promoter, a Na_(v)1.7 promoter,a Na_(v)1.8 promoter, a Na_(v)1.9 promoter, or an Advillin promoter). Insome embodiments, the tissue specific promoter is a muscle-specificpromoter (e.g., a creatine kinase (MCK) promoter). In some embodiments,the promoter is a methyl CpG binding protein 2 (MeCP2) promoter or aderivative thereof (e.g., a MeCP2 promoter truncated to about 229 bpand/or a MeCP2 promoter truncated to about 406 bp). In some embodiments,the promoter comprises an intronic sequence. In some embodiments, thepromoter comprises a bidirectional promoter and/or an enhancer (e.g., aCMV enhancer).

In some embodiments, the polynucleotide further encodes a dosageindicator protein. In some embodiments, the dosage indicator protein,the payload protein, and the protease are expressed as a fusion protein.In some embodiments, the nucleic acid comprises a secondary promoteroperably linked to a secondary polynucleotide encoding a dosageindicator protein. In some embodiments, the promoter and the secondarypromoter are different. In some embodiments, the dosage indicatorprotein is detectable. In some embodiments, the dosage indicator proteincomprises green fluorescent protein (GFP), enhanced green fluorescentprotein (EGFP), yellow fluorescent protein (YFP), enhanced yellowfluorescent protein (EYFP), blue fluorescent protein (BFP), redfluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry,mruby3 rsCherry, rsCherryRev, derivatives thereof, or any combinationthereof.

In some embodiments, one or more cells comprise an endogenous version ofthe payload gene, and wherein the promoter comprises or is derived fromthe promoter of the endogenous version. In some embodiments, the payloadprotein comprises a disease-associated protein, wherein aberrantexpression of the disease-associated protein correlates with theoccurrence and/or progression of the disease.

In some embodiments, the payload protein comprises a protein associatedwith an expression-sensitive disease or disorder. In some embodiments,the payload protein comprises methyl CpG binding protein 2 (MeCP2),DRK1A, KAT6A, NIPBL, HDAC4, UBE3A, EHMT1, one or more genes encoded onchromosome 9q34.3, NPHP1, LIMK1 one or more genes encoded on chromosome7q11.23, P53, TPI1, FGFR1 and related genes, RA1, SHANK3, CLN3, NF-1,TP53, PFK, CD40L, CYP19A1, PGRN, CHRNA7, PMP22, CD40LG, derivativesthereof, or any combination thereof.

In some embodiments, the payload protein comprises fluorescenceactivity, polymerase activity, protease activity, phosphatase activity,kinase activity, SUMOylating activity, deSUMOylating activity,ribosylation activity, deribosylation activity, myristoylation activitydemyristoylation activity, or any combination thereof. In someembodiments, the payload protein comprises nuclease activity,methyltransferase activity, demethylase activity, DNA repair activity,DNA damage activity, deamination activity, dismutase activity,alkylation activity, depurination activity, oxidation activity,pyrimidine dimer forming activity, integrase activity, transposaseactivity, recombinase activity, polymerase activity, ligase activity,helicase activity, photolyase activity, glycosylase activity,acetyltransferase activity, deacetylase activity, adenylation activity,deadenylation activity, or any combination thereof.

In some embodiments, the payload protein comprises a CRE recombinase,GCaMP, a cell therapy component, a knock-down gene therapy component, acell-surface exposed epitope, or any combination thereof. In someembodiments, the payload protein comprises a chimeric antigen receptor.In some embodiments, the payload protein is associated with anagricultural trait of interest selected from the group consisting ofincreased yield, increased abiotic stress tolerance, increased droughttolerance, increased flood tolerance, increased heat tolerance,increased cold and frost tolerance, increased salt tolerance, increasedheavy metal tolerance, increased low-nitrogen tolerance, increaseddisease resistance, increased pest resistance, increased herbicideresistance, increased biomass production, male sterility, or anycombination thereof. In some embodiments, the payload protein isassociated with a biological manufacturing process selected from thegroup comprising fermentation, distillation, biofuel production,production of a compound, production of a polypeptide, or anycombination thereof.

In some embodiments, the payload protein comprises a diagnostic agent.In some embodiments, the diagnostic agent comprises green fluorescentprotein (GFP), enhanced green fluorescent protein (EGFP), yellowfluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP),blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP,Dronpa, Padron, mApple, mCherry, mruby3, rsCherry, rsCherryRev,derivatives thereof, or any combination thereof. In some embodiments,the payload protein comprises a nuclear localization signal (NLS) or anuclear export signal (NES).

In some embodiments, the payload protein comprises a programmablenuclease. In some embodiments, the programmable nuclease is selectedfrom the group comprising: SpCas9 or a derivative thereof, VRER, VQR,EQR SpCas9; xCas9-3.7; eSpCas9; Cas9-HF1; HypaCas9; evoCas9; HiFi Cas9;ScCas9; StCas9; NmCas9; SaCas9; CjCas9; CasX; Cas9 H940A nickase; Cas12and derivatives thereof, dcas9-APOBEC1 fusion, BE3, and dcas9-deaminasefusions; dcas9-Krab, dCas9-VP64, dCas9-Tet1, and dcas9-transcriptionalregulator fusions; Dcas9-fluorescent protein fusions; Cas13-fluorescentprotein fusions; RCas9-fluorescent protein fusions; Cas13-adenosinedeaminase fusions. In some embodiments, the programmable nucleasecomprises a zinc finger nuclease (ZFN) and/or transcriptionactivator-like effector nuclease (TALEN). In some embodiments, theprogrammable nuclease comprises Streptococcus pyogenes Cas9 (SpCas9),Staphylococcus aureus Cas9 (SaCas9), a zinc finger nuclease, TALeffector nuclease, meganuclease, MegaTAL, Tev-m TALEN, MegaTev, homingendonuclease, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8,Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e,Cas13a, Cas13b, Cas13c, derivatives thereof, or any combination thereof.In some embodiments, the nucleic acid further comprises a polynucleotideencoding (i) a targeting molecule and/or (ii) a donor nucleic acid. Insome embodiments, the targeting molecule is capable of associating withthe programmable nuclease. In some embodiments, the targeting moleculecomprises single strand DNA or single strand RNA. In some embodiments,the targeting molecule comprises a single guide RNA (sgRNA).

In some embodiments, the polynucleotide further encodes one or moresecondary proteins. In some embodiments, the payload protein and the oneor more secondary proteins are expressed as a fusion protein. In someembodiments, the 3′UTR of the transgene(s) encoding the one or moresecondary proteins comprises one or more silencer effector bindingsequences. In some embodiments, the payload protein and the one or moresecondary proteins comprise a synthetic protein circuit.

In some embodiments, the nucleic acid is a vector (e.g., a viral vector,a plasmid, a naked DNA vector, a lipid nanoparticle, or any combinationthereof). In some embodiments, the viral vector is an AAV vector, alentivirus vector, a retrovirus vector, an integration-deficientlentivirus (IDLV) vector.

Disclosed herein include compositions comprising a nucleic aciddisclosed herein. In some embodiments, the composition comprises avector, a ribonucleoprotein (RNP) complex, a liposome, a nanoparticle,an exosome, a microvesicle, or any combination thereof, comprising anucleic acid disclosed herein.

In some embodiments, the composition comprises (i) a targeting moleculeor a nucleic acid encoding the targeting molecule and/or (ii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid. In someembodiments, the targeting molecule is capable of associating with theprogrammable nuclease. In some embodiments, the targeting moleculecomprises single strand DNA or single strand RNA. In some embodiments,the targeting molecule comprises a single guide RNA (sgRNA).

In some embodiments, the vector is a viral vector, a plasmid, a nakedDNA vector, a lipid nanoparticle, or any combination thereof. In someembodiments, the viral vector is an AAV vector, a lentivirus vector, aretrovirus vector, an integration-deficient lentivirus (IDLV) vector. Insome embodiments, the AAV vector comprises single-stranded AAV (ssAAV)vector or a self-complementary AAV (scAAV) vector. In some embodiments,the AAV vector comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, derivatives thereof, or any combination thereof. In someembodiments, the AAV vector comprises an AAV9 variant engineered forsystemic delivery (e.g., AAV-PHP.B, AAV-PHP.eB, or AAV-PHP.S). In someembodiments, the vector is a neurotropic viral vector (e.g., comprisesor is derived from Herpesviridae, varicella zoster virus, pseudorabiesvirus, cyromegalovirus, Epstein-barr virus, encephalitis virus, poliovirus, coxsackie virus, echo virus, mumps virus, measles virus, rabiesvirus, or any combination thereof).

Disclosed herein include methods of treating a disease or disorder in asubject. In some embodiments, the method comprises: introducing into oneor more cells of a subject in need thereof a composition comprising anucleic acid disclosed herein, a composition disclosed herein, and/or anucleic acid disclosed herein.

Disclosed herein include methods for tuned dosage-invariant expressionof a payload protein in one or more cells. In some embodiments, themethod comprises: introducing into one or more cells a compositioncomprising a nucleic acid disclosed herein, a composition disclosedherein, and/or a nucleic acid disclosed herein.

In some embodiments, the one or more cells comprise one or more cells ofa subject. In some embodiments, the subject is suffering from a diseaseor disorder. In some embodiments, the one or more cells comprise aneuron. In some embodiments, the neuron is associated with aneurological disease or disorder. The method can comprise: introducingan inducer (e.g., doxycycline) of the inducible promoter to the one ormore cells. In some embodiments, the introducing step comprisesadministering an initial dose of the inducer followed one or more lowermaintenance doses of the inducer.

In some embodiments, the introducing step comprises administering acomposition comprising a nucleic acid disclosed herein, a compositiondisclosed herein, and/or a nucleic acid disclosed herein, to a subjectcomprising the one or more cells. In some embodiments, the administeringcomprises systemic administration (e.g., intravenous, intramuscular,intraperitoneal, or intraarticular). In some embodiments, administeringcomprises intrathecal administration, intracranial injection, aerosoldelivery, nasal delivery, vaginal delivery, rectal delivery, buccaldelivery, ocular delivery, local delivery, topical delivery,intracisternal delivery, intraperitoneal delivery, oral delivery,intramuscular injection, intravenous injection, subcutaneous injection,intranodal injection, intratumoral injection, intraperitoneal injection,intradermal injection, or any combination thereof.

In some embodiments, administering comprises an injection into a brainregion (e.g., direct administration to the brain parenchyma). In someembodiments, the brain region comprises the Lateral parabrachialnucleus, brainstem, Medulla oblongata, Medullary pyramids, Olivary body,Inferior olivary nucleus, Rostral ventrolateral medulla, Respiratorycenter, Dorsal respiratory group, Ventral respiratory group,Pre-Botzinger complex, Botzinger complex, Paramedian reticular nucleus,Cuneate nucleus, Gracile nucleus, Intercalated nucleus, Area postrema,Medullary cranial nerve nuclei, Inferior salivatory nucleus, Nucleusambiguus, Dorsal nucleus of vagus nerve, Hypoglossal nucleus, Solitarynucleus, Pons, Pontine nuclei, Pontine cranial nerve nuclei, chief orpontine nucleus of the trigeminal nerve sensory nucleus (V), Motornucleus for the trigeminal nerve (V), Abducens nucleus (VI), Facialnerve nucleus (VII), vestibulocochlear nuclei (vestibular nuclei andcochlear nuclei) (VIII), Superior salivatory nucleus, Pontine tegmentum,Respiratory centers, Pneumotaxic center, Apneustic center, Pontinemicturition center (Barrington's nucleus), Locus coeruleus,Pedunculopontine nucleus, Laterodorsal tegmental nucleus, Tegmentalpontine reticular nucleus, Superior olivary complex, Paramedian pontinereticular formation, Cerebellar peduncles, Superior cerebellar peduncle,Middle cerebellar peduncle, Inferior cerebellar peduncle, Cerebellum,Cerebellar vermis, Cerebellar hemispheres, Anterior lobe, Posteriorlobe, Flocculonodular lobe, Cerebellar nuclei, Fastigial nucleus,Interposed nucleus, Globose nucleus, Emboliform nucleus, Dentatenucleus, Tectum, Corpora quadrigemina, inferior colliculi, superiorcolliculi, Pretectum, Tegmentum, Periaqueductal gray, Parabrachial area,Medial parabrachial nucleus, Subparabrachial nucleus (Kolliker-Fusenucleus), Rostral interstitial nucleus of medial longitudinalfasciculus, Midbrain reticular formation, Dorsal raphe nucleus, Rednucleus, Ventral tegmental area, Substantia nigra, Pars compacta, Parsreticulata, Interpeduncular nucleus, Cerebral peduncle, Crus cerebri,Mesencephalic cranial nerve nuclei, Oculomotor nucleus (III), Trochlearnucleus (IV), Mesencephalic duct (cerebral aqueduct, aqueduct ofSylvius), Pineal body, Habenular nucleim Stria medullares, Taeniathalami, Subcommissural organ, Thalamus, Anterior nuclear group,Anteroventral nucleus (aka ventral anterior nucleus), Anterodorsalnucleus, Anteromedial nucleus, Medial nuclear group, Medial dorsalnucleus, Midline nuclear group, Paratenial nucleus, Reuniens nucleus,Rhomboidal nucleus, Intralaminar nuclear group, Centromedial nucleus,Parafascicular nucleus, Paracentral nucleus, Central lateral nucleus,Central medial nucleus, Lateral nuclear group, Lateral dorsal nucleus,Lateral posterior nucleus, Pulvinar, Ventral nuclear group, Ventralanterior nucleus, Ventral lateral nucleus, Ventral posterior nucleus,Ventral posterior lateral nucleus, Ventral posterior medial nucleus,Metathalamus, Medial geniculate body, Lateral geniculate body, Thalamicreticular nucleus, Hypothalamus, limbic system, HPA axis, preoptic area,Medial preoptic nucleus, Suprachiasmatic nucleus, Paraventricularnucleus, Supraoptic nucleusm Anterior hypothalamic nucleus, Lateralpreoptic nucleus, median preoptic nucleus, periventricular preopticnucleus, Tuberal, Dorsomedial hypothalamic nucleus, Ventromedialnucleus, Arcuate nucleus, Lateral area, Tuberal part of Lateral nucleus,Lateral tuberal nuclei, Mammillary nuclei, Posterior nucleus, Lateralarea, Optic chiasm, Subfornical organ, Periventricular nucleus,Pituitary stalk, Tuber cinereum, Tuberal nucleus, Tuberomammillarynucleus, Tuberal region, Mammillary bodies, Mammillary nucleus,Subthalamus, Subthalamic nucleus, Zona incerta, Pituitary gland,neurohypophysis, Pars intermedia, adenohypophysis, cerebral hemispheres,Corona radiata, Internal capsule, External capsule, Extreme capsule,Arcuate fasciculus, Uncinate fasciculus, Perforant Path, Hippocampus,Dentate gyms, Cornu ammonis, Cornu ammonis area 1, Cornu ammonis area 2,Cornu ammonis area 3, Cornu ammonis area 4, Amygdala, Central nucleus,Medial nucleus (accessory olfactory system), Cortical and basomedialnuclei, Lateral and basolateral nuclei, extended amygdala, Striaterminalis, Bed nucleus of the stria terminalis, Claustrum, Basalganglia, Striatum, Dorsal striatum (aka neostriatum), Putamen, Caudatenucleus, Ventral striatum, Striatum, Nucleus accumbens, Olfactorytubercle, Globus pallidus, Subthalamic nucleus, Basal forebrain,Anterior perforated substance, Substantia innominata, Nucleus basalis,Diagonal band of Broca, Septal nuclei, Medial septal nuclei, Laminaterminalis, Vascular organ of lamina terminalis, Olfactory bulb,Piriform cortex, Anterior olfactory nucleus, Olfactory tract, Anteriorcommissure, Uncus, Cerebral cortex, Frontal lobe, Frontal cortex,Primary motor cortex, Supplementary motor cortex, Premotor cortex,Prefrontal cortex, frontopolar cortex, Orbitofrontal cortex,Dorsolateral prefrontal cortex, dorsomedial prefrontal cortex,ventrolateral prefrontal cortex, Superior frontal gyms, Middle frontalgyms, Inferior frontal gyms, Brodmann areas (4, 6, 8, 9, 10, 11, 12, 24,25, 32, 33, 44, 45, 46, and/or 47), Parietal lobe, Parietal cortex,Primary somatosensory cortex (S1), Secondary somatosensory cortex (S2),Posterior parietal cortex, postcentral gyms, precuneus, Brodmann areas(1, 2, 3 (Primary somesthetic area), 5, 7, 23, 26, 29, 31, 39, and/or40), Occipital lobe, Primary visual cortex (V1), V2, V3, V4, V5/MT,Lateral occipital gyms, Cuneus, Brodmann areas (17 (V1, primary visualcortex), 18, and/or 19), temporal lobe, Primary auditory cortex (A1),secondary auditory cortex (A2), Inferior temporal cortex, Posteriorinferior temporal cortex, Superior temporal gyms, Middle temporal gyms,Inferior temporal gyms, Entorhinal Cortex, Perirhinal Cortex,Parahippocampal gyms, Fusiform gyms, Brodmann areas (9, 20, 21, 22, 27,34, 35, 36, 37, 38, 41, and/or 42), Medial superior temporal area (MST),insular cortex, cingulate cortex, Anterior cingulate, Posteriorcingulate, dorsal cingulate, Retrosplenial cortex, Indusium griseum,Subgenual area 25, Brodmann areas (23, 24; 26, 29, 30 (retrosplenialareas), 31, and/or 32), cranial nerves (Olfactory (I), Optic (II),Oculomotor (III), Trochlear (IV), Trigeminal (V), Abducens (VI), Facial(VII), Vestibulocochlear (VIII), Glossopharyngeal (IX), Vagus (X),Accessory (XI), Hypoglossal (XII)), or any combination thereof.

In some embodiments, the brain region comprises neural pathways Superiorlongitudinal fasciculus, Arcuate fasciculus, Thalamocortical radiations,Cerebral peduncle, Corpus callosum, Posterior commissure, Pyramidal orcorticospinal tract, Medial longitudinal fasciculus, dopamine system,Mesocortical pathway, Mesolimbic pathway, Nigrostriatal pathway,Tuberoinfundibular pathway, serotonin system, Norepinephrine Pathways,Posterior column-medial lemniscus pathway, Spinothalamic tract, Lateralspinothalamic tract, Anterior spinothalamic tract, or any combinationthereof.

The method can comprise: isolating the one or more cells from thesubject prior to the introducing step and/or comprising administeringthe one or more cells into a subject after the introducing step. In someembodiments, the introducing step is performed in vivo, in vitro, and/orex vivo. In some embodiments, the introducing step comprises calciumphosphate transfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, electrical nucleartransport, chemical transduction, electrotransduction,Lipofectamine-mediated transfection, Effectene-mediated transfection,lipid nanoparticle (LNP)-mediated transfection, or any combinationthereof.

In some embodiments, the one or more cells comprise a eukaryotic cell.In some embodiments, the eukaryotic cell comprises an antigen-presentingcell, a dendritic cell, a macrophage, a neural cell, a brain cell, anastrocyte, a microglial cell, and a neuron, a spleen cell, a lymphoidcell, a lung cell, a lung epithelial cell, a skin cell, a keratinocyte,an endothelial cell, an alveolar cell, an alveolar macrophage, analveolar pneumocyte, a vascular endothelial cell, a mesenchymal cell, anepithelial cell, a colonic epithelial cell, a hematopoietic cell, a bonemarrow cell, a Claudius cell, Hensen cell, Merkel cell, Muller cell,Paneth cell, Purkinje cell, Schwann cell, Sertoli cell, acidophil cell,acinar cell, adipoblast, adipocyte, brown or white alpha cell, amacrinecell, beta cell, capsular cell, cementocyte, chief cell, chondroblast,chondrocyte, chromaffin cell, chromophobic cell, corticotroph, deltacell, Langerhans cell, follicular dendritic cell, enterochromaffin cell,ependymocyte, epithelial cell, basal cell, squamous cell, endothelialcell, transitional cell, erythroblast, erythrocyte, fibroblast,fibrocyte, follicular cell, germ cell, gamete, ovum, spermatozoon,oocyte, primary oocyte, secondary oocyte, spermatid, spermatocyte,primary spermatocyte, secondary spermatocyte, germinal epithelium, giantcell, glial cell, astroblast, astrocyte, oligodendroblast,oligodendrocyte, glioblast, goblet cell, gonadotroph, granulosa cell,haemocytoblast, hair cell, hepatoblast, hepatocyte, hyalocyte,interstitial cell, juxtaglomerular cell, keratinocyte, keratocyte,lemmal cell, leukocyte, granulocyte, basophil, eosinophil, neutrophil,lymphoblast, B-lymphoblast, T-lymphoblast, lymphocyte, B-lymphocyte,T-lymphocyte, helper induced T-lymphocyte, Th1 T-lymphocyte, Th2T-lymphocyte, natural killer cell, thymocyte, macrophage, Kupffer cell,alveolar macrophage, foam cell, histiocyte, luteal cell, lymphocyticstem cell, lymphoid cell, lymphoid stem cell, macroglial cell,mammotroph, mast cell, medulloblast, megakaryoblast, megakaryocyte,melanoblast, melanocyte, mesangial cell, mesothelial cell,metamyelocyte, monoblast, monocyte, mucous neck cell, myoblast, myocyte,muscle cell, cardiac muscle cell, skeletal muscle cell, smooth musclecell, myelocyte, myeloid cell, myeloid stem cell, myoblast,myoepithelial cell, myofibrobast, neuroblast, neuroepithelial cell,neuron, odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell,parafollicular cell, paraluteal cell, peptic cell, pericyte, peripheralblood mononuclear cell, phaeochromocyte, phalangeal cell, pinealocyte,pituicyte, plasma cell, platelet, podocyte, proerythroblast,promonocyte, promyeloblast, promyelocyte, pronormoblast, reticulocyte,retinal pigment epithelial cell, retinoblast, small cell, somatotroph,stem cell, sustentacular cell, teloglial cell, a zymogenic cell, or anycombination thereof. In some embodiments, the stem cell comprises anembryonic stem cell, an induced pluripotent stem cell (iPSC), ahematopoietic stem/progenitor cell (HSPC), or any combination thereof.In some embodiments, the one or more cells comprise two or more cells.In some embodiments, the two or more cells comprise a first cell typeand a second cell type.

In some embodiments, the polynucleotide is transcribed at a rate atleast 1.1-fold higher in the first cell type as compared to the secondcell type. In some embodiments, the steady state levels of the payloadtranscript are at least 1.1-fold higher in the first cell type ascompared to the second cell type. In some embodiments, the rate oftranscription of the polynucleotide and/or the rate of translation ofthe payload transcript varies between a first time point and a secondtime point in a single cell and/or varies between the first cell typeand the second cell type at the same time point. In some embodiments, inthe absence of the protease and/or the silencer effector, the payloadprotein reaches untuned steady state payload protein levels in the oneor more cells. In some embodiments, untuned steady state payload proteinlevels range between a lower untuned threshold and an upper untunedthreshold of an untuned expression range. In some embodiments, steadystate dosage indicator protein levels reflect untuned steady statepayload protein levels. In some embodiments, in the presence theprotease and/or the silencer effector, the payload protein reaches tunedsteady state payload protein levels in the one or more cells. In someembodiments, tuned steady state payload protein levels range between alower tuned threshold and an upper tuned threshold of a tuned expressionrange. In some embodiments, at the first time point and the second timepoint in a single cell, the steady state levels of the payload proteinremain within the tuned expression range. In some embodiments, in thefirst cell type and the second cell type at the same time point, thesteady state levels of the payload protein remain within the tunedexpression range.

In some embodiments, the lower tuned threshold and/or the upper tunedthreshold of a tuned expression range can be reduced by increasing thenumber of silencer effector binding sequences in the 3′ UTR and/orincreasing the degree of complementarity between the silencer effectorand the one or more silencer effector binding sequences. In someembodiments, the lower tuned threshold and/or the upper tuned thresholdof the tuned expression range can be increased by reducing the number ofsilencer effector binding sequences in the 3′ UTR and/or reducing thedegree of complementarity between the silencer effector and the one ormore silencer effector binding sequences. In some embodiments, the lowertuned threshold and/or the upper tuned threshold of a tuned expressionrange can be increased by introducing one or more non-canonical aminoacid substitutions into the cut site. In some embodiments, the lowertuned threshold and/or the upper tuned threshold of a tuned expressionrange can be reduced by introducing one or more canonical amino acidsubstitutions into the cut site.

In some embodiments, the difference between the lower untuned thresholdand the upper untuned threshold of the untuned expression range isgreater than about two orders of magnitude. In some embodiments, thedifference between the lower tuned threshold and the upper tunedthreshold of the tuned expression range is less than about one order ofmagnitude. In some embodiments, the payload protein is efficacious atsteady state payload protein levels within the tuned expression range.In some embodiments, the payload protein is inefficacious and/or toxicat steady state payload protein levels above and/or below the tunedexpression range. In some embodiments, the payload protein is capable ofinducing an immunogenic response and/or a cytokine storm at steady statepayload protein levels outside the tuned expression range. In someembodiments, tuned steady state payload protein levels comprise atherapeutic level of the payload protein. In some embodiments, thesteady state payload protein levels remain within the tuned expressionrange across multiple cell types, titers of viral vector, and/or viralvector capsid types. In some embodiments, the tuned steady state payloadprotein levels are robust to tissue tropism and stochastic expression.

In some embodiments, the disease or disorder comprises a MECP2-relateddisorder selected from the group comprising Classic Rett Syndrome,MECP2-related Severe Neonatal Encephalopathy, PPM-X Syndrome, or anycombination thereof. In some embodiments, the disease or disorder is ablood disease, an immune disease, a cancer, an infectious disease, agenetic disease, a disorder caused by aberrant mtDNA, a metabolicdisease, a disorder caused by aberrant cell cycle, a disorder caused byaberrant angiogenesis, a disorder cause by aberrant DNA damage repair,or any combination thereof. In some embodiments, the disease or disordercomprises a neurological disease or disorder. In some embodiments, theneurological disease or disorder comprises Alzheimer's disease,Creutzfeld-Jakob's syndrome/disease, bovine spongiform encephalopathy(BSE), prion related infections, diseases involving mitochondrialdysfunction, diseases involving β-amyloid and/or tauopathy, Down'ssyndrome, hepatic encephalopathy, Huntington's disease, motor neurondiseases, amyotrophic lateral sclerosis (ALS), olivoponto-cerebellaratrophy, post-operative cognitive deficit (POCD), systemic lupuserythematosus, systemic clerosis, Sjogren's syndrome, Neuronal CeroidLipofuscinosis, neurodegenerative cerebellar ataxias, Parkinson'sdisease, Parkinson's dementia, mild cognitive impairment, cognitivedeficits in various forms of mild cognitive impairment, cognitivedeficits in various forms of dementia, dementia pugilistica, vascularand frontal lobe dementia, cognitive impairment, learning impairment,eye injuries, eye diseases, eye disorders, glaucoma, retinopathy,macular degeneration, head or brain or spinal cord injuries, head orbrain or spinal cord trauma, convulsions, epileptic convulsions,epilepsy, temporal lobe epilepsy, myoclonic epilepsy, tinnitus,dyskinesias, chorea, Huntington's chorea, athetosis, dystonia,stereotypy, ballism, tardive dyskinesias, tic disorder, torticollisspasmodicus, blepharospasm, focal and generalized dystonia, nystagmus,hereditary cerebellar ataxias, corticobasal degeneration, tremor,essential tremor, addiction, anxiety disorders, panic disorders, socialanxiety disorder (SAD), attention deficit hyperactivity disorder (ADHD),attention deficit syndrome (ADS), restless leg syndrome (RLS),hyperactivity in children, autism, dementia, dementia in Alzheimer'sdisease, dementia in Korsakoff syndrome, Korsakoff syndrome, vasculardementia, dementia related to HIV infections, HIV-1 encephalopathy, AIDSencephalopathy, AIDS dementia complex, AIDS-related dementia, majordepressive disorder, major depression, depression, memory loss, stress,bipolar manic-depressive disorder, drug tolerance, drug tolerance toopioids, movement disorders, fragile-X syndrome, irritable bowelsyndrome (IBS), migraine, multiple sclerosis (MS), muscle spasms, pain,chronic pain, acute pain, inflammatory pain, neuropathic pain,posttraumatic stress disorder (PTSD), schizophrenia, spasticity,Tourette's syndrome, eating disorders, food addiction, binge eatingdisorders, agoraphobia, generalized anxiety disorder,obsessive-compulsive disorder, panic disorder, social phobia, phobicdisorders, substance-induced anxiety disorder, delusional disorder,schizoaffective disorder, schizophreniform disorder, substance-inducedpsychotic disorder, hypertension, or any combination thereof.

In some embodiments, the disease or disorder is an expression-sensitivedisease or disorder. In some embodiments, the expression-sensitivedisease or disorder is selected from the group comprising Rett Syndrome;Smith-Magenis Syndrome; Phelan-McDermid Syndrome; Cornelia de LangeSyndrome and other NIPBL related disorders; DRK1A, KAT6A and relateddisorders of severe intellectual disability; Chromosome 2Q37 DeletionSyndrome and other HDAC4 Related Disorders; Angelman Syndrome; KleefstraSyndrome; Joubert Syndrome and other NPHP1 Related Disorders; WilliamsSyndrome; Neurofibromatosis Type 1; Li-Fraumeni syndrome and similarp53-related cancer syndromes; Phosphofructokinase Deficiency; X-linkedHyper IgM Syndrome and similar primary immunodeficiency disorders;Triosephosphate isomerase deficiency; Kallman Syndrome; AromataseDeficiency; Batten Disease, Frontotemporal Dementia and otherneurodegenerative disorders related to loss of progranulin; CholinergicReceptor Nicotinic Alpha 7 Subunit Related Disorders; and HereditaryNeuropathy with liability to Pressure Palsies.

In some embodiments, an expression-sensitive disease or disorder ischaracterized by decreased expression of one or more proteins, whereinectopic overexpression of said one or more proteins at a steady statelevel beyond the upper tuned threshold causes cellular toxicity and/ordisease. In some embodiments, said expression-sensitive disorder is aneurodevelopmental syndromic disorder. In some embodiments, theneurodevelopmental syndromic disorder is selected from the groupcomprising Rett Syndrome, Smith-Magenis Syndrome (RA1), Phelan-McDermidSyndrome (SHANK3), Cornelia de Lange Syndrome (NIPBL) and other NIPBLrelated disorders, DRK1A, KAT6A and related disorders of severeintellectual disability, Chromosome 2Q37 Deletion Syndrome and otherHDAC4 Related Disorders, Angelman Syndrome, Kleefstra Syndrome, JoubertSyndrome and other NPHP1 Related Disorders, and Williams Syndrome. Insome embodiments, said expression-sensitive disorder is a proliferativedisorder and/or cancer. In some embodiments, the proliferative disorderand/or cancer is selected from the group comprising NeurofibromatosisType 1 and Li-Fraumeni syndrome and similar p53-related cancersyndromes. In some embodiments, said expression-sensitive disorder is aglycogen storage disorder. In some embodiments, the glycogen storagedisorder is phosphofructokinase deficiency. In some embodiments, saidexpression-sensitive disorder is a hematologic disorder and/or immunedisorder. In some embodiments, the hematologic disorder and/or immunedisorder is selected from the group comprising X-linked Hyper IgMSyndrome and related primary immunodeficiency disorders, andtriosephosphate isomerase deficiency. In some embodiments, saidexpression-sensitive disorder is an endocrine disorder. In someembodiments, the endocrine disorder is selected from the groupcomprising Kallman Syndrome and Aromatase Deficiency. In someembodiments, said expression-sensitive disorder is a neuropsychiatricdisorder. In some embodiments, the neuropsychiatric disorder is selectedfrom the group comprising Batten Disease, Frontotemporal Dementia andother neurodegenerative disorders related to loss of progranulin,Cholinergic Receptor Nicotinic Alpha 7 Subunit Related Disorders, andHereditary Neuropathy with liability to Pressure Palsies.

In some embodiments, the payload protein comprises RA1 and the diseaseor disorder comprises Smith-Magenis Syndrome. In some embodiments, thepayload protein comprises SHANK3 and the disease or disorder comprisesPhelan-McDermid Syndrome. In some embodiments, the payload proteincomprises CLN3 and the disease or disorder comprises Batten Disease. Insome embodiments, the payload protein comprises NF-1 and the disease ordisorder comprises Neurofibromitosis Type I. In some embodiments, thepayload protein comprises TP53 and the disease or disorder comprisesLi-Fraumeni Syndrome. In some embodiments, the payload protein comprisesPFK and the disease or disorder comprises phosphofructokinasedeficiency. In some embodiments, the payload protein comprises CD40LGand the disease or disorder comprises X-linked Hyper IGM disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E depict non-limiting exemplary embodiments and data relatedto a single robust protein control circuit using TEVP as protease. FIG.1A illustrates a non-limiting exemplary single transcript protein-levelIFFL provided herein. FIG. 1B depicts fluorescence microscopy imagesshowing de-correlated input and output as a result of the IFFL. FIG. 1Cdepicts exemplary data showing that tunability is established usingdifferent amino acid sequences. FIG. 1D depicts a non-limiting exemplarynumerical simulation of the steady state gene expression. FIG. 1Edepicts flow cytometry data confirming the numerical and analytic modelsof TEVP performance.

FIGS. 2A-2B depict non-limiting exemplary embodiments and data relatedto IFFL constructs provided herein. FIG. 2A illustrates non-limitingexemplary single transcript protein-level IFFLs provided herein. FIG. 2Bdepicts flow cytometry data. Error bars indicate ±1 standard deviationin log space.

FIGS. 3A-3B depict non-limiting exemplary embodiments and data relatedto packing of an IFFL construct into AAV vectors. FIG. 3B depicts flowcytometry data. Dots represent medians of bins. Error bars indicate ±1standard deviation in log space.

FIGS. 4A-4B depict non-limiting exemplary embodiments and data relatedto synthetic miRNA IFFL circuits. FIG. 4A illustrates non-limitingexemplary miRNA-level IFFLs provided herein. FIG. 4B depicts flowcytometry data. Dots represent medians of bins. Error bars indicate ±1standard deviation in log space.

FIGS. 5A-5B depict non-limiting exemplary embodiments and data relatedto synthetic miRNA IFFL circuits. FIG. 5A illustrates non-limitingexemplary miRNA-level IFFLs provided herein. FIG. 5B depicts flowcytometry data showing 4 levels of MeCP2-EGFP expression. Dots representmedians of bins. Error bars indicate ±1 standard deviation in log space.

FIGS. 6A-6B depict non-limiting exemplary embodiments and data relatedto an IFFL circuit motif modulating the expression of virally deliveredcargo in mammals.

FIGS. 7A-7B depict non-limiting exemplary embodiments and data relatedto a protein-level IFFL driven by the CMV promoter.

FIGS. 8A-8B depict non-limiting exemplary embodiments and data relatedto a protein-level IFFL driven by the full Ef1a promoter (with intron).

FIGS. 9A-9B depict non-limiting exemplary embodiments and data relatedto a protein-level IFFL driven by Ef1a promoter (without the intron).

FIGS. 10A-10B depict non-limiting exemplary embodiments and data relatedto a first embodiment of a miRNA-level IFFL circuit.

FIGS. 11A-11B depict non-limiting exemplary embodiments and data relatedto a second embodiment of a miRNA-level IFFL circuit.

FIGS. 12A-12B depict non-limiting exemplary embodiments and data relatedto a third embodiment of a miRNA-level IFFL circuit.

FIG. 13 illustrates a non-limiting exemplary illustration of amiRNA-level IFFL circuit engineered to reduce endogenous MeCP2expression.

FIGS. 14A-14C depict non-limiting exemplary code and simulations.

FIGS. 15A-15B depict non-limiting exemplary code and simulations.

FIGS. 16A-16B depict non-limiting exemplary code and simulations.

FIGS. 17A-17B depict non-limiting exemplary code and simulations.

FIGS. 18A-18B depict non-limiting exemplary code and simulations.

FIGS. 19A-19B depict non-limiting exemplary code and simulations.

FIGS. 20A-20C depict non-limiting exemplary code and simulations.

FIGS. 21A-21B depict non-limiting exemplary code and simulations.

FIGS. 22A-22B depict non-limiting exemplary code and simulations.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe Figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein and made part of the disclosure herein.

All patents, published patent applications, other publications, andsequences from GenBank, and other databases referred to herein areincorporated by reference in their entirety with respect to the relatedtechnology.

Disclosed herein include nucleic acids. In some embodiments, the nucleicacid comprises: a promoter operably linked to a polynucleotide encodinga fusion protein comprising a payload protein, a protease, and one ormore self-cleaving peptide sequences, wherein the payload proteincomprises a degron and a cut site the protease is capable of cutting toexpose the degron, and wherein the degron of the payload protein beingexposed changes the payload protein to a payload protein destabilizedstate. In some embodiments, the promoter is capable of inducing thetranscription of the polynucleotide to generate a payload transcript.

Disclosed herein include nucleic acids. In some embodiments, the nucleicacid comprises: a promoter operably linked to a polynucleotidecomprising a payload gene and a silencer effector cassette, wherein thepayload gene 3′UTR comprises one or more silencer effector bindingsequences. In some embodiments, the promoter is capable of inducing thetranscription of the polynucleotide to generate a payload transcript. Insome embodiments, the payload gene encodes a payload protein. In someembodiments, payload gene encodes a siRNA, a shRNA, an antisense RNAoligonucleotide, an antisense miRNA, a trans-splicing RNA, a guide RNA,single-guide RNA, crRNA, a tracrRNA, a trans-splicing RNA, a pre-mRNA, amRNA, or any combination thereof. In some embodiments, the silencereffector cassette comprises a miRNA cassette. The polynucleotide cancomprise: a transcript stabilization element (e.g., hepatitispost-translational regulatory element (WPRE), bovine growth hormonepolyadenylation (bGH-polyA) signal sequence, human growth hormonepolyadenylation (hGH-polyA) signal sequence, or any combinationthereof).

Disclosed herein include compositions comprising a nucleic aciddisclosed herein. In some embodiments, the composition comprises avector, a ribonucleoprotein (RNP) complex, a liposome, a nanoparticle,an exosome, a microvesicle, or any combination thereof, comprising anucleic acid disclosed herein.

Disclosed herein include methods of treating a disease or disorder in asubject. In some embodiments, the method comprises: introducing into oneor more cells of a subject in need thereof a composition comprising anucleic acid disclosed herein, a composition disclosed herein, and/or anucleic acid disclosed herein.

Disclosed herein include methods for tuned dosage-invariant expressionof a payload protein in one or more cells. In some embodiments, themethod comprises: introducing into one or more cells a compositioncomprising a nucleic acid disclosed herein, a composition disclosedherein, and/or a nucleic acid disclosed herein.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. See, e.g. Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley& Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.1989). For purposes of the present disclosure, the following terms aredefined below.

As used herein, the term “vector” refers to a polynucleotide construct,typically a plasmid or a virus, used to transmit genetic material to ahost cell (e.g., a target cell). Vectors can be, for example, viruses,plasmids, cosmids, or phage. A vector can be a viral vector. A vector asused herein can be composed of either DNA or RNA. In some embodiments, avector is composed of DNA. An “expression vector” is a vector that iscapable of directing the expression of a protein encoded by one or moregenes carried by the vector when it is present in the appropriateenvironment. Vectors are preferably capable of autonomous replication.Typically, an expression vector comprises a transcription promoter, agene, and a transcription terminator. Gene expression is usually placedunder the control of a promoter, and a gene is said to be “operablylinked to” the promoter.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide (e.g., a synthetic proteincircuit component) from nucleic acid sequences contained therein linkedto transcriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g. 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “operably linked” is used to describe theconnection between regulatory elements and a gene or its coding region.Typically, gene expression is placed under the control of one or moreregulatory elements, for example, without limitation, constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.A gene or coding region is said to be “operably linked to” or“operatively linked to” or “operably associated with” the regulatoryelements, meaning that the gene or coding region is controlled orinfluenced by the regulatory element. For instance, a promoter isoperably linked to a coding sequence if the promoter effectstranscription or expression of the coding sequence.

The term “construct,” as used herein, refers to a recombinant nucleicacid that has been generated for the purpose of the expression of aspecific nucleotide sequence(s), or that is to be used in theconstruction of other recombinant nucleotide sequences.

As used herein, the terms “nucleic acid” and “polynucleotide” areinterchangeable and refer to any nucleic acid, whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sultone linkages, andcombinations of such linkages. The terms “nucleic acid” and“polynucleotide” also specifically include nucleic acids composed ofbases other than the five biologically occurring bases (adenine,guanine, thymine, cytosine and uracil).

The term “regulatory element” and “expression control element” are usedinterchangeably and refer to nucleic acid molecules that can influencethe expression of an operably linked coding sequence in a particularhost organism. These terms are used broadly to and cover all elementsthat promote or regulate transcription, including promoters, coreelements required for basic interaction of RNA polymerase andtranscription factors, upstream elements, enhancers, and responseelements (see, e.g., Lewin, “Genes V” (Oxford University Press, Oxford)pages 847-873). Exemplary regulatory elements in prokaryotes includepromoters, operator sequences and a ribosome binding sites. Regulatoryelements that are used in eukaryotic cells can include, withoutlimitation, transcriptional and translational control sequences, such aspromoters, enhancers, splicing signals, polyadenylation signals,terminators, protein degradation signals, internal ribosome-entryelement (IRES), 2A sequences, and the like, that provide for and/orregulate expression of a coding sequence and/or production of an encodedpolypeptide in a host cell.

As used herein, 2A sequences or elements refer to small peptidesintroduced as a linker between two proteins, allowing autonomousintraribosomal self-processing of polyproteins (See e.g., de Felipe.Genetic Vaccines and Ther. 2: 13 (2004); deFelipe et al. Traffic5:616-626 (2004)). These short peptides allow co-expression of multipleproteins from a single vector. Many 2A elements are known in the art.Examples of 2A sequences that can be used in the methods and systemdisclosed herein, without limitation, include 2A sequences from thefoot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A),Thosea asigna virus (T2A), and porcine tescho virus-1 (P2A).

As used herein, the term “promoter” is a nucleotide sequence thatpermits binding of RNA polymerase and directs the transcription of agene. Typically, a promoter is located in the 5′ non-coding region of agene, proximal to the transcriptional start site of the gene. Sequenceelements within promoters that function in the initiation oftranscription are often characterized by consensus nucleotide sequences.Examples of promoters include, but are not limited to, promoters frombacteria, yeast, plants, viruses, and mammals (including humans). Apromoter can be inducible, repressible, and/or constitutive. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asa change in temperature.

As used herein, the term “enhancer” refers to a type of regulatoryelement that can increase the efficiency of transcription, regardless ofthe distance or orientation of the enhancer relative to the start siteof transcription.

As used herein, the term “variant” refers to a polynucleotide (orpolypeptide) having a sequence substantially similar to a referencepolynucleotide (or polypeptide). In the case of a polynucleotide, avariant can have deletions, substitutions, additions of one or morenucleotides at the 5′ end, 3′ end, and/or one or more internal sites incomparison to the reference polynucleotide. Similarities and/ordifferences in sequences between a variant and the referencepolynucleotide can be detected using conventional techniques known inthe art, for example polymerase chain reaction (PCR) and hybridizationtechniques. Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis. Generally, a variant of a polynucleotide,including, but not limited to, a DNA, can have at least about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99% or more sequence identity to thereference polynucleotide as determined by sequence alignment programsknown by skilled artisans. In the case of a polypeptide, a variant canhave deletions, substitutions, additions of one or more amino acids incomparison to the reference polypeptide. Similarities and/or differencesin sequences between a variant and the reference polypeptide can bedetected using conventional techniques known in the art, for exampleWestern blot. Generally, a variant of a polypeptide, can have at leastabout 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99% or more sequence identity to thereference polypeptide as determined by sequence alignment programs knownby skilled artisans.

As used herein, the term “effective amount” refers to an amountsufficient to effect beneficial or desirable biological and/or clinicalresults.

As used herein, a “subject” refers to an animal that is the object oftreatment, observation or experiment. “Animal” includes cold- andwarm-blooded vertebrates and invertebrates such as fish, shellfish,reptiles, and in particular, mammals. “Mammal,” as used herein, refersto an individual belonging to the class Mammalia and includes, but notlimited to, humans, domestic and farm animals, zoo animals, sports andpet animals. Non-limiting examples of mammals include mice; rats;rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates,such as monkeys, chimpanzees and apes, and, in particular, humans. Insome embodiments, the mammal is a human. However, in some embodiments,the mammal is not a human.

As used herein, the term “treatment” refers to an intervention made inresponse to a disease, disorder or physiological condition manifested bya patient. The aim of treatment may include, but is not limited to, oneor more of the alleviation or prevention of symptoms, slowing orstopping the progression or worsening of a disease, disorder, orcondition and the remission of the disease, disorder or condition. Theterm “treat” and “treatment” includes, for example, therapeutictreatments, prophylactic treatments, and applications in which onereduces the risk that a subject will develop a disorder or other riskfactor. Treatment does not require the complete curing of a disorder andencompasses embodiments in which one reduces symptoms or underlying riskfactors. In some embodiments, “treatment” refers to both therapeutictreatment and prophylactic or preventative measures. Those in need oftreatment include those already affected by a disease or disorder orundesired physiological condition as well as those in which the diseaseor disorder or undesired physiological condition is to be prevented. Forexample, in some embodiments treatment may reduce the level of RASsignaling in the subject, thereby to reduce, alleviate, or eradicate thesymptom(s) of the disease(s). As used herein, the term “prevention”refers to any activity that reduces the burden of the individual laterexpressing those symptoms. This can take place at primary, secondaryand/or tertiary prevention levels, wherein: a) primary prevention avoidsthe development of symptoms/disorder/condition; b) secondary preventionactivities are aimed at early stages of the condition/disorder/symptomtreatment, thereby increasing opportunities for interventions to preventprogression of the condition/disorder/symptom and emergence of symptoms;and c) tertiary prevention reduces the negative impact of an alreadyestablished condition/disorder/symptom by, for example, restoringfunction and/or reducing any condition/disorder/symptom or relatedcomplications. The term “prevent” does not require the 100% eliminationof the possibility of an event. Rather, it denotes that the likelihoodof the occurrence of the event has been reduced in the presence of thecompound or method.

“Pharmaceutically acceptable” carriers are ones which are nontoxic tothe cell or mammal being exposed thereto at the dosages andconcentrations employed. “Pharmaceutically acceptable” carriers can be,but not limited to, organic or inorganic, solid or liquid excipientswhich is suitable for the selected mode of application such as oralapplication or injection, and administered in the form of a conventionalpharmaceutical preparation, such as solid such as tablets, granules,powders, capsules, and liquid such as solution, emulsion, suspension andthe like. Often the physiologically acceptable carrier is an aqueous pHbuffered solution such as phosphate buffer or citrate buffer. Thephysiologically acceptable carrier may also comprise one or more of thefollowing: antioxidants including ascorbic acid, low molecular weight(less than about 10 residues) polypeptides, proteins, such as serumalbumin, gelatin, immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone, amino acids, carbohydrates including glucose,mannose, or dextrins, chelating agents such as EDTA, sugar alcohols suchas mannitol or sorbitol, salt-forming counterions such as sodium, andnonionic surfactants such as Tween, polyethylene glycol (PEG), andPluronics. Auxiliary, stabilizer, emulsifier, lubricant, binder, pHadjuster controller, isotonic agent and other conventional additives mayalso be added to the carriers.

Robust and Tunable Control of Gene Expression

There are provided, in some embodiments, methods, compositions, andsystems for robust and tunable control of expression of a payload (e.g.,payload protein, payload gene). There are provided, in some embodiments,rationally designed circuits enabling the precise control of payloadexpression (e.g., MeCP2 expression) required for gene therapy (e.g.,Rett Syndrome gene therapy). The compositions and methods providedherein enable a tunable and robust control of payload gene expressionthat solves the problems of standard gene therapy vectors and methods,including but, not limited to, variation in gene delivery due to tropismor accessibility; stochasticity in transcription, translation, or otheraspects of expression; and heterogeneity in the levels of cellularcomponents that affect protein expression. For example, embodimentsprovided wherein the payload protein comprises MeCP2 display bothnon-toxic expression of MeCP2 in the liver and therapeuticallysufficient MeCP2 expression in the brain. There are provided, in someembodiments, rationally designed circuits that control the expression ofits payload, including miRNA-level and protein-level incoherentfeed-forward loop circuits. In some embodiments, said circuits providetunable control of gene expression in order to set the expression of anectopic gene (e.g., transgene, payload protein, payload gene) to atherapeutic level, robust to tissue tropism and stochastic expression.The compositions (e.g., circuits) and methods provided herein solve theproblem of MeCP2 expression control in Rett Syndrome gene therapy andcan be broadly applied to gene and cell therapies, biologicalmanufacturing processes, and/or any application wherein proteinexpression stabilization is useful or necessary.

Incoherent feedforward loops have been shown to provide adaptiveexpression levels, but have not been implemented synthetically withtunable control, at the protein level, or demonstrated as safe andeffective in a gene therapy. The incoherent feed-forward loop (IFFL) wasfirst identified as a highly conserved biological circuit motif in E.coli. The motif was later shown to be one of the only biological circuitarchitectures that maintains a robust steady state over wide ranges ofinput. Synthetic incoherent feedforward loops have been implemented tostabilize expression of molecular sensors, to adapt gene expression tochanges in gene dosage received during transient transfection (Bleris,Leonidas, et al. “Synthetic incoherent feedforward circuits showadaptation to the amount of their genetic template.” Molecular systemsbiology 7.1 (2011): 519) and to buffer gene expression against noise atthe single copy level. Currently available compositions and methodsemploying IFFLs fail to address the existing problems in the art, as ithas not been shown that these circuits are tunable, that they can adaptexpression over 3 orders of dosage magnitude, that they can beimplemented at the protein-level, or that they are safe and effective inan in-vivo gene therapy.

The compositions and methods provided herein can be applied to genetherapies, cell therapies, or cell line development for biologicalmanufacturing. In some embodiments, the circuits provided herein can beemployed to express MeCP2 at a controlled level for Rett Syndrome genetherapy. However, the applications of these circuits extend to any genetherapy, in particular gene therapies wherein expression must becontrolled, and many other applications in cell therapy or biologicalmanufacturing. For example, in CAR T-cell therapy, the activation ofdesigned T-cells depends on the level of chimeric antigen receptor (CAR)expressed. Noise in CAR expression, for example due to the lentiviralintegration site, is propagated through to noise in T-cell activationand increases off target activation. The IFFLs provided herein canstabilize CAR expression and reduce off-target activation. Moreover,this application is not be limited to CARs; for example, the IFFLsprovided herein can stabilize expression of sensors used in celltherapy. In some embodiments, the methods and compositions providedherein mitigate Cas9 genotoxicity: Cas9 off-target effects are known tooccur and would decrease if Cas9 is expressed at a lower level. Finally,many biological manufacturing processes must deal with varyingconditions, such as pH, that result in changes of enzymatic pathwaycomponent expression and can thereby result in low yields. IFFLsprovided herein can stabilize the expression of these pathway componentsto reduce yield variability. The catalytic IFFL modules disclosed hereincan improve gene therapies, cell based therapies, and biologicalmanufacturing processes. Because the provided methods and compositionscan solve a general problem—achieving precisely tunable gene expressionlevels—the constructs provided herein can a standard enabling componentfor a wide range of existing and emerging biomedical applications.Provided herein is the first instance of IFFL-based compositions andmethods being used in a gene therapy, first instance of being shown tobe tunable, and first instance of being shown to have multiple order ofmagnitude regulation.

Disclosed herein include nucleic acids. In some embodiments, the nucleicacid comprises: a promoter operably linked to a polynucleotide encodinga fusion protein comprising a payload protein, a protease, and one ormore self-cleaving peptide sequences. The payload protein can comprise adegron and a cut site the protease is capable of cutting to expose thedegron. The degron of the payload protein being exposed can change thepayload protein to a payload protein destabilized state. The promotercan be capable of inducing the transcription of the polynucleotide togenerate a payload transcript. The payload transcript can encode thefusion protein. A payload gene can encode the payload transcript (e.g.,the fusion protein). The nucleic acid can be a vector (e.g., a viralvector, a plasmid, a naked DNA vector, a lipid nanoparticle, or anycombination thereof). The nucleic acid can be an expression vector. Theviral vector can be an AAV vector, a lentivirus vector, a retrovirusvector, an integration-deficient lentivirus (IDLV) vector.

As described herein, a “cut site” is a peptide sequence specific for oneor more proteases that when recognized or bound by the one or moreproteases are cleaved by the one or more proteases. The peptide sequenceof the cut site may be specific for one protease or a type of proteases,or may be general to multiple proteases or types of proteases.

As used herein, “destabilize” may refer to the ability of a peptide ormolecule to prevent or stop the same or another molecule or peptide frommaintaining a particular state. “Destabilize” may also refer to theability of a peptide or molecule to allow or increase the amount ofdegradation that the same or another molecule or peptide faces, such asby increasing the affinity of the same or other molecule or peptide to adigestive protein.

The degron can comprise an N-degron. Some degrons areubiquitin-dependent or ubiquitin-independent. The self-cleaving peptidesequence can comprise porcine teschovirus-1 2A peptide (P2A), Thoseaasigna virus 2A peptide (T2A), equine rhinitis A virus 2A peptide (E2A),foot-and-mouth disease virus 2A peptide (F2A), or any combinationthereof. The protease can comprise tobacco etch virus (TEV) protease,tobacco vein mottling virus (TVMV) protease, hepatitis C virus protease(HCVP), derivatives thereof, or any combination thereof. The proteasecan comprise TEVP. The cut site can comprise an amino acid sequence atleast about 25% (e.g., 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, ora number or a range between any two of these values) homologous to thecanonical cut of site of a protease. The cut site can comprise an aminoacid sequence at least about 25% homologous to the amino acid sequenceof ETVFFQ (SEQ ID NO: 1), ENAYFQ (SEQ ID NO: 2), ENLFFQ (SEQ ID NO: 3),ENLYFQ (SEQ ID NO: 4), ENLYFQY (SEQ ID NO: 5), ENLYFQF (SEQ ID NO: 6),ENLYFQQ (SEQ ID NO: 7), or ENLFFQY (SEQ ID NO: 8).

There are provided, in some embodiments, degrons. A degron can compriseDI-FR degron, an N-degron, a phospho degron, a heat inducible degron, aphotosensitive degron, an oxygen dependent degron, ornithinedecarboxylase degron, estrogen receptor domain degrons, a ecDHFR degron,an FKBP degron, a UnaG degron, or any combination thereof. As anon-limiting example, the degron may be an ornithine decarboxylasedegron. The degron can comprise a ecDHFR degron. In some embodiments,the degron and/or cut site is located at the 5′ end of the payloadprotein. In some embodiments, the degron and/or cut site is located atthe 3′ end of the payload protein. In some embodiments, the degronand/or cut site situated within the payload protein. In someembodiments, the degron and/or cut site situated within the payloadprotein to for a split protein. In some embodiments, the sequence of thedegron (e.g., N-end degron) is varied to tune the expression of thepayload protein.

Disclosed herein include nucleic acids. In some embodiments, the nucleicacid comprises: a promoter operably linked to a polynucleotidecomprising a payload gene and a silencer effector cassette. The payloadgene can comprises one or more silencer effector binding sequences. Theone or more silencer effector binding sequences can be located anywherein the payload gene, such as the 3′UTR, 5′UTR, intron, or anycombination thereof. The promoter can be capable of inducing thetranscription of the polynucleotide to generate a payload transcript. Insome embodiments, the payload gene encodes a payload protein. In someembodiments, the payload gene encodes a siRNA, a shRNA, an antisense RNAoligonucleotide, an antisense miRNA, a trans-splicing RNA, a guide RNA,single-guide RNA, crRNA, a tracrRNA, a trans-splicing RNA, a pre-mRNA, amRNA, or any combination thereof, which can target the expression of anyendogenous gene of interest. The polynucleotide can comprise: atranscript stabilization element (e.g., hepatitis post-translationalregulatory element (WPRE), bovine growth hormone polyadenylation(bGH-polyA) signal sequence, human growth hormone polyadenylation(hGH-polyA) signal sequence, or any combination thereof).

The nucleic acid can be a vector (e.g., a viral vector, a plasmid, anaked DNA vector, a lipid nanoparticle, or any combination thereof). Theviral vector can be an AAV vector, a lentivirus vector, a retrovirusvector, an integration-deficient lentivirus (IDLV) vector.

The silencer effector cassette can comprise a miRNA cassette. An introncan be located in the payload gene 3′UTR, payload gene 5′UTR, or betweenpayload gene exons. The intron can comprise the silencer effectorcassette. The intron can comprise: (i) an intronic insert encoding asilencer effector, (ii) a donor splice site, (iii) an acceptor splicesite, (iv) a branch point domain; and/or (v) a polypyrimidine tract. Thesilencer effector can be capable of being released from said intron byan intron excision mechanism selected from the group comprising cellularRNA splicing and/or processing machinery, nonsense-mediated decay (NMD)processing, or any combination thereof. The silencer effector cancomprise a microRNA (miRNA), a precursor microRNA (pre-miRNA), a smallinterfering RNA (siRNA), a short-hairpin RNA (shRNA), precursorsthereof, derivatives thereof, or a combination thereof.

The silencer effector can be capable of binding the one or more silencereffector binding sequences, thereby reducing the stability of thepayload transcript and/or reducing the translation of the payloadtranscript. The one or more silencer effector binding sequences cancomprise miRNA binding sites. The polynucleotide can comprise about 1 toabout 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or a number or arange between any two of these values) silencer binding sequences. Theone or more silencer effector binding sequences can be about 1 to about50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any twoof these values) nucleotides in length. The silencer effector cancomprise a region of complementarity that is complementary with at least5 consecutive nucleotides of the one or more silencer effector bindingsequences. The silencer effector can comprise at least about 25% (e.g.,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or a number or a range between any two of thesevalues) complementarity to the one or more silencer effector bindingsequences. In some embodiments, one or more cells comprise an endogenousversion (e.g., variant) of a gene encoding the payload protein, andwherein the silencer effector can comprise at least about 25% (e.g.,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or a number or a range between any two of thesevalues) complementarity to one or more endogenous silencer effectorbinding sequences within the 3′UTR of the endogenous version. In someembodiments, one or more cells comprise an endogenous version (e.g.,variant) of a gene encoding the payload protein comprising one or moresecondary silencer binding sequences in the 3′UTR, wherein the nucleicacid can comprise a secondary silencer effector cassette encoding asecondary silencer effector that is capable of binding the one or moresecondary silencer binding sequences. In some embodiments, the payloadprotein is expressed and the endogenous version (e.g., variant) of thepayload protein is not expressed.

Disclosed herein include compositions comprising a nucleic aciddisclosed herein. In some embodiments, the composition comprises avector, a ribonucleoprotein (RNP) complex, a liposome, a nanoparticle,an exosome, a microvesicle, or any combination thereof, comprising anucleic acid disclosed herein. The vector can be a viral vector, aplasmid, a naked DNA vector, a lipid nanoparticle, or any combinationthereof. The viral vector can be an AAV vector, a lentivirus vector, aretrovirus vector, an integration-deficient lentivirus (IDLV) vector.The AAV vector can comprise single-stranded AAV (ssAAV) vector or aself-complementary AAV (scAAV) vector. The AAV vector can comprise AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, derivatives thereof, orany combination thereof. The AAV vector can comprise an AAV9 variantengineered for systemic delivery (e.g., AAV-PHP.B, AAV-PHP.eB, orAAV-PHP.S). The vector can be a neurotropic viral vector (e.g.,comprises or derived from Herpesviridae, varicella zoster virus,pseudorabies virus, cyromegalovirus, Epstein-barr virus, encephalitisvirus, polio virus, coxsackie virus, echo virus, mumps virus, measlesvirus, rabies virus, or any combination thereof).

Disclosed herein include methods of treating a disease or disorder in asubject. Disclosed herein include methods of preventing a disease ordisorder in a subject. Disclosed herein include methods of diagnosing adisease or disorder in a subject. Disclosed herein include methods ofperforming cell therapy. Disclosed herein include methods of performingbiological manufacturing. In some embodiments, the method comprises:introducing into one or more cells of a subject in need thereof acomposition comprising a nucleic acid disclosed herein, a compositiondisclosed herein, and/or a nucleic acid disclosed herein. In someembodiments, the payload protein is a wild-type version of an endogenousprotein. In some embodiments, the payload protein is a variant of anendogenous protein.

Disclosed herein include methods for tuned dosage-invariant expressionof a payload protein in one or more cells. In some embodiments, themethod comprises: introducing into one or more cells a compositioncomprising a nucleic acid disclosed herein, a composition disclosedherein, and/or a nucleic acid disclosed herein. The one or more cellscan comprise one or more cells of a subject. The subject can besuffering from a disease or disorder. The one or more cells can comprisea neuron, such as, for example, a neuron associated with a neurologicaldisease or disorder.

In some embodiments, the introducing step comprises administering acomposition comprising a nucleic acid disclosed herein, a compositiondisclosed herein, and/or a nucleic acid disclosed herein, to a subjectcomprising the one or more cells. The method can comprise: introducingan inducer (e.g., doxycycline) of the inducible promoter to the one ormore cells. The introducing step can comprise administering an initialdose of the inducer followed one or more lower maintenance doses of theinducer.

The method can comprise: isolating the one or more cells from thesubject prior to the introducing step and/or comprising administeringthe one or more cells into a subject after the introducing step. Theintroducing step can be performed in vivo, in vitro, and/or ex vivo. Theintroducing step can comprise calcium phosphate transfection,DEAE-dextran mediated transfection, cationic lipid-mediatedtransfection, electroporation, electrical nuclear transport, chemicaltransduction, electrotransduction, Lipofectamine-mediated transfection,Effectene-mediated transfection, lipid nanoparticle (LNP)-mediatedtransfection, or any combination thereof.

The one or more cells can comprise a eukaryotic cell. The eukaryoticcell can comprise an antigen-presenting cell, a dendritic cell, amacrophage, a neural cell, a brain cell, an astrocyte, a microglialcell, and a neuron, a spleen cell, a lymphoid cell, a lung cell, a lungepithelial cell, a skin cell, a keratinocyte, an endothelial cell, analveolar cell, an alveolar macrophage, an alveolar pneumocyte, avascular endothelial cell, a mesenchymal cell, an epithelial cell, acolonic epithelial cell, a hematopoietic cell, a bone marrow cell, aClaudius cell, Hensen cell, Merkel cell, Muller cell, Paneth cell,Purkinje cell, Schwann cell, Sertoli cell, acidophil cell, acinar cell,adipoblast, adipocyte, brown or white alpha cell, amacrine cell, betacell, capsular cell, cementocyte, chief cell, chondroblast, chondrocyte,chromaffin cell, chromophobic cell, corticotroph, delta cell, Langerhanscell, follicular dendritic cell, enterochromaffin cell, ependymocyte,epithelial cell, basal cell, squamous cell, endothelial cell,transitional cell, erythroblast, erythrocyte, fibroblast, fibrocyte,follicular cell, germ cell, gamete, ovum, spermatozoon, oocyte, primaryoocyte, secondary oocyte, spermatid, spermatocyte, primary spermatocyte,secondary spermatocyte, germinal epithelium, giant cell, glial cell,astroblast, astrocyte, oligodendroblast, oligodendrocyte, glioblast,goblet cell, gonadotroph, granulosa cell, haemocytoblast, hair cell,hepatoblast, hepatocyte, hyalocyte, interstitial cell, juxtaglomerularcell, keratinocyte, keratocyte, lemmal cell, leukocyte, granulocyte,basophil, eosinophil, neutrophil, lymphoblast, B-lymphoblast,T-lymphoblast, lymphocyte, B-lymphocyte, T-lymphocyte, helper inducedT-lymphocyte, Th1 T-lymphocyte, Th2 T-lymphocyte, natural killer cell,thymocyte, macrophage, Kupffer cell, alveolar macrophage, foam cell,histiocyte, luteal cell, lymphocytic stem cell, lymphoid cell, lymphoidstem cell, macroglial cell, mammotroph, mast cell, medulloblast,megakaryoblast, megakaryocyte, melanoblast, melanocyte, mesangial cell,mesothelial cell, metamyelocyte, monoblast, monocyte, mucous neck cell,myoblast, myocyte, muscle cell, cardiac muscle cell, skeletal musclecell, smooth muscle cell, myelocyte, myeloid cell, myeloid stem cell,myoblast, myoepithelial cell, myofibrobast, neuroblast, neuroepithelialcell, neuron, odontoblast, osteoblast, osteoclast, osteocyte, oxynticcell, parafollicular cell, paraluteal cell, peptic cell, pericyte,peripheral blood mononuclear cell, phaeochromocyte, phalangeal cell,pinealocyte, pituicyte, plasma cell, platelet, podocyte,proerythroblast, promonocyte, promyeloblast, promyelocyte,pronormoblast, reticulocyte, retinal pigment epithelial cell,retinoblast, small cell, somatotroph, stem cell, sustentacular cell,teloglial cell, a zymogenic cell, or any combination thereof. The stemcell can comprise an embryonic stem cell, an induced pluripotent stemcell (iPSC), a hematopoietic stem/progenitor cell (HSPC), or anycombination thereof.

The one or more cells can comprise two or more cells (e.g., two or morecells of a subject). The subject can comprise a plurality of cells.Administering the nucleic acids, compositions, and/or vectors providedherein to a subject can cause expression of the payload in two or morecells of the subject. The two or more cells can comprise 2 or more(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 100000, or a numberor a range between any two of these values) different cell types. Samecell types and/or different cell types can be located in different areasof a subject. The basal transcriptional and/or translational machineryof the different cell types can vary amongst themselves and each other.The transcription of the payload transcript and/or translation of thepayload transcript can vary among different cells. The two or more cellscan comprise a first cell type and a second cell type. The first celltype and second cell type can be the same or different. Thepolynucleotide can be transcribed at a rate at least 1.1-fold (e.g.,1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold,1000-fold, 10000-fold, or a number or a range between any of thesevalues) higher in the first cell type as compared to the second celltype. The steady state levels of the payload transcript can be at least1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 1000-fold, 10000-fold, or a number or a range between any ofthese values) higher in the first cell type as compared to the secondcell type.

In some embodiments, the polynucleotide further encodes a dosageindicator protein. The dosage indicator protein, the payload protein,and the protease can be expressed as a fusion protein. The nucleic acidcan comprise a secondary promoter operably linked to a secondarypolynucleotide encoding a dosage indicator protein. The promoter and thesecondary promoter can be different. The dosage indicator protein can bedetectable. The dosage indicator protein can comprise green fluorescentprotein (GFP), enhanced green fluorescent protein (EGFP), yellowfluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP),blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP,Dronpa, Padron, mApple, mCherry, mruby3, rsCherry, rsCherryRev,derivatives thereof, or any combination thereof. In some embodiments,steady state dosage indicator protein levels reflect untuned steadystate payload protein levels.

In some embodiments, the rate of transcription of the polynucleotideand/or the rate of translation of the payload transcript varies betweena first time point and a second time point in a single cell and/orvaries between the first cell type and the second cell type at the sametime point. In some embodiments, in the absence of the protease and/orthe silencer effector, the payload protein reaches untuned steady statepayload protein levels in the one or more cells. In some embodiments,untuned steady state payload protein levels range between a loweruntuned threshold and an upper untuned threshold of an untunedexpression range. In some embodiments, in the presence the proteaseand/or the silencer effector, the payload protein reaches tuned steadystate payload protein levels in the one or more cells. In someembodiments, tuned steady state payload protein levels range between alower tuned threshold and an upper tuned threshold of a tuned expressionrange. In some embodiments, at the first time point and the second timepoint in a single cell, the steady state levels of the payload proteinremain within the tuned expression range. In some embodiments, in thefirst cell type and the second cell type at the same time point, thesteady state levels of the payload protein remain within the tunedexpression range.

In some embodiments, the lower tuned threshold and/or the upper tunedthreshold of a tuned expression range can be reduced at least 1.1-fold(e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold,1000-fold, 10000-fold, or a number or a range between any of thesevalues) by increasing the number of silencer effector binding sequencesin the 3′ UTR and/or increasing the degree of complementarity betweenthe silencer effector and the one or more silencer effector bindingsequences. In some embodiments, the lower tuned threshold and/or theupper tuned threshold of the tuned expression range can be increased atleast 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 1000-fold, 10000-fold, or a number or a range between any ofthese values) by reducing the number of silencer effector bindingsequences in the 3′ UTR and/or reducing the degree of complementaritybetween the silencer effector and the one or more silencer effectorbinding sequences.

Adjusting the sequence of the cut site to more or less closelyapproximate the canonical cut site of the protease can alter the lowertuned threshold and/or the upper tuned threshold of a tuned expressionrange. In some embodiments, the lower tuned threshold and/or the uppertuned threshold of a tuned expression range can be increased at least1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 1000-fold, 10000-fold, or a number or a range between any ofthese values) by introducing one or more non-canonical amino acidsubstitutions into the cut site. In some embodiments, the lower tunedthreshold and/or the upper tuned threshold of a tuned expression rangecan be reduced at least 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold,1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, 1000-fold, 10000-fold, or a numberor a range between any of these values) by introducing one or morecanonical amino acid substitutions into the cut site.

In some embodiments, the difference between the lower untuned thresholdand the upper untuned threshold of the untuned expression range isgreater than about two orders of magnitude. The difference between thelower tuned threshold and the upper tuned threshold of the tunedexpression range can be less than about one order of magnitude. Thedifference between the lower tuned threshold and the upper tunedthreshold of the tuned expression range can be less than about 1.1-fold(e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold,1000-fold, 10000-fold, or a number or a range between any of thesevalues) than the difference between the lower untuned threshold and theupper untuned threshold of the untuned expression range. In someembodiments, less than about 0.1%, less than about 1%, less than about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%,or a number or a range between any two of these values, of the cells ofa subject comprising have steady-state levels of the payload proteinoutside of the tuned expression range. The period of time between theintroducing and/or administering steps and the cell reachingsteady-state levels of the payload protein can be about 100000 hours,10000 hours, 1000 hours, 100 hours, 50 hours, 24 hours, 8 hours, about 7hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about2 hours, about 1 hour, about 30 minutes, about 15 minutes, about 10minutes, about 5 minutes, about 1 minute, or a number or a range betweenany two of these values.

The payload protein can be efficacious at steady state payload proteinlevels within the tuned expression range. The payload protein can beinefficacious and/or toxic at steady state payload protein levels aboveand/or below the tuned expression range. The payload protein can becapable of inducing an immunogenic response and/or a cytokine storm atsteady state payload protein levels outside the tuned expression range.Tuned steady state payload protein levels can comprise a therapeuticlevel of the payload protein. In some embodiments, the steady statepayload protein levels remain within the tuned expression range acrossmultiple cell types, titers of viral vector, and/or viral vector capsidtypes. The tuned steady state payload protein levels can be robust totissue tropism and stochastic expression. In some embodiments of thecompositions and methods, the circuits provided herein are not preciselydosage invariant but are useful to providing reduced dosage dependenceand/or limiting the levels of protein expression. For example, in someembodiments, the upper tuned threshold can be at least about 1.1-fold(e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold,1000-fold, 10000-fold, or a number or a range between any of thesevalues) lower than the upper untuned threshold.

There are provided, in some embodiments, methods and compositions forself-regulated gene therapy that can provide regulated expressionindependent of gene dosage. In some embodiments, a self-regulated genetherapy payload can provide regulated expression of a payload (e.g.,MeCP2) independent of gene dosage. In some embodiments, the methods andcompositions provide dosage invariance of a payload. Dosage invariance,as used herein, shall be given its ordinary meaning, and shall alsorefer to a lower fold change in protein output compared to gene dosage.The methods and compositions provided herein can yield and at least1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 1000-fold, 10000-fold, or a number or a range between any ofthese values) lower fold change in protein output as compared to genedosage. In some embodiments, the methods and compositions provided hereenable payload dosage invariance in vivo (e.g., in a mouse, in a human).

Some embodiments of the methods and compositions provided hereincomprise a protein-level implementation. In some embodiments, theprotein-level implementation comprises the following constructstructure: [degron]-[Gene (e.g.,payload)]-[T2A]-[protease]-[P2a]-[dosage indicator protein (e.g.,mCherry)]. In some embodiments, the methods and compositions providedhere enable tuning of the expression level of a payload by varyingprotease cleavage site sequence. The disclosed methods and compositionscan function across a broad range of useful promoters, such as, but notlimited to, CMV, Ef1a, and Ef1a (no intron) promoters. In someembodiments, the methods and composition operates identically indifferent species of cells, using different capsid variants, and/or atdifferent viral titers.

Some embodiments of the methods and compositions provided hereincomprise a miRNA-level implementation. In some embodiments, themiRNA-level implementation comprises the following construct structure:[Gene (e.g., payload)]-[miRNAtarget]-[miRNAcasette]. In someembodiments, the methods and compositions provided here enable tuning ofthe expression level of a payload by varying the number of miRNA targetsites included in the construct. The construct can function acrossdiverse promoters. (e.g., the same behavior across 3 promoter variants).Some embodiments of the methods and compositions enable dosage invariantgene product replacement, by including additional miRNA, on the sameconstruct, directed specifically against the endogenous but not theectopic gene copy. Some embodiments provided herein related to thecombination of the miRNA-level and protein-level circuits providedherein (e.g., a fusion protein encoded by a transcript that alsocomprises miRNA binding sites and a miRNA cassette).

Some embodiments of the method and compositions provided herein comprisedosage-invariant MeCP2 gene therapy for Rett Syndrome. In someembodiments, the methods and compositions comprise a viral vector (e.g.AAV vectors, including but not limited to, AAV9 and AAV9 engineeredvariants for systemic delivery (e.g., AAV.PHP.eB)). The payload cancomprise any gene for dosage-invariant delivery of dosage-sensitive geneproducts for gene therapy applications, including, but not limited to,RA1 for Smith-Magenis Syndrome; SHANK3 for Phelan-McDermid Syndrome;CLN3 for Batten Disease; NF-1 for Neurofibromitosis Type I; TP53 forLi-Fraumeni Syndrome; PFK for phosphofructokinase deficiency; and/orCD40LG for X-linked Hyper IGM disorders. Some embodiments of the methodand compositions provided herein comprise a payload for treating aneurologic disease or disorder, glycogen storage disorders, hematologicdisease or disorder, and any other diseases or disorders that aresubject to a so-called “goldilocks” problem, wherein the delivered gene(e.g., transgene, payload) must be carefully regulated within a certainrange. In some embodiments, the compositions are delivered viacerebrospinal fluid and/or CSF routes.

Some embodiments of the method and compositions provided herein comprisegene therapy applications wherein the immunogenicity of a payload needsto be reduced by avoiding unnecessary overexpression. Some embodimentscomprise cell therapies (e.g., CAR-T cell therapies), wherein themethods and compositions herein enable uniform regulation of chimericantigen receptor (CAR) expression as well as other cell therapycomponents in order to improve specificity and/or reduce toxicity (e.g.,cytokine storm). Some embodiments of the disclosed method andcompositions reduce, prevent and/or avoid overexpression toxicity ingene therapies based on protein expression. Some embodiments of thedisclosed method and compositions reduce, prevent and/or avoid potentialtoxicity from overexpression of components in knock-down gene therapies.The provided method and compositions can comprise regulating CRErecombinase to reduce CRE-related toxicity, regulating GCaMP to reduceGCaMP-related toxicity, and/or regulating dCas9 to reduce dcas9 relatedgenotoxicity. In some embodiments, the disclosed systems provide thesame output for both ssAAV and scAAV vectors, even though the overallexpression of the circuit may change. Embodiments of the methods andcompositions provided herein can comprise single-stranded (ss) andself-complementary (sc) genomes. In some embodiments, the cargo can becomprise ss and/or sc cargo. The disclosed systems can be inserted intolentiviral constructs, or other viral vectors. The promoter can compriseone or more of the following: PGK, 3-phosphoglycerate kinase promoter,SV40 virus promoter; RSV virus promoter; CBA/HBA, chicken/human betaactin promoters; CMV virus promoter; UBC, ubiquitin C promoter; CAG;CBH; TRE/Tre3G; Gadph; Ef1a, elongation factor 1 alpha promoter; MeP229,MeCP2 promoter, truncated to 229 bp; MeP406, MeCP2 promoter, truncatedto 406 bp; SYN, synapsin (human/otherwise) promoter; CamKIIa promoter;and MCK. The promoter can comprise one or more gene therapy promotersknown in the art. In some embodiments, the functional form of theexpression remains the same with single stranded AAV (ss) and selfcomplementary AAV (sc) vectors. In some embodiments, the use of AAV (sc)vectors result in higher overall expression.

In some embodiments, the payload comprises a gene that has shown or issuspected of presenting immunogenicity issues when ectopicallyexpressed. In some embodiments, the payload regulated by the systemsprovided herein is an immunogenic gene product, and can include, forexample, genes provided in Table 1 (e.g., Batten's Disease/FTD). Someexamples of the disclosed methods and compositions include regulatingexpression in antigen presenting cells and regulating expression ofcell-surface exposed epitopes. In some embodiments, the methods comprisesystemic delivery for the nervous system (e.g., for treatment of Rettsyndrome) wherein the system can used differentially to reduceexpression in pb organs such as the liver (liver toxicity) or the heart(cardiac arrest). In some embodiments of the methods and compositionsdisclosed herein, side-effects of systemic gene therapy (e.g., payloadoverexpression in heart causing cardiac arrest and/or payloadoverexpression in liver causing liver toxicity) are prevented by thetunable and dosage-invariant compositions and methods provided herein.

The disease or disorder can comprise a MECP2-related disorder selectedfrom the group comprising Classic Rett Syndrome, MECP2-related SevereNeonatal Encephalopathy, PPM-X Syndrome, or any combination thereof. Thedisease or disorder can be a blood disease, an immune disease, a cancer,an infectious disease, a genetic disease, a disorder caused by aberrantmtDNA, a metabolic disease, a disorder caused by aberrant cell cycle, adisorder caused by aberrant angiogenesis, a disorder cause by aberrantDNA damage repair, or any combination thereof.

The disease or disorder can comprise a neurological disease or disorder.The neurological disease or disorder can comprise Alzheimer's disease,Creutzfeld-Jakob's syndrome/disease, bovine spongiform encephalopathy(BSE), prion related infections, diseases involving mitochondrialdysfunction, diseases involving β-amyloid and/or tauopathy, Down'ssyndrome, hepatic encephalopathy, Huntington's disease, motor neurondiseases, amyotrophic lateral sclerosis (ALS), olivoponto-cerebellaratrophy, post-operative cognitive deficit (POCD), systemic lupuserythematosus, systemic clerosis, Sjogren's syndrome, Neuronal CeroidLipofuscinosis, neurodegenerative cerebellar ataxias, Parkinson'sdisease, Parkinson's dementia, mild cognitive impairment, cognitivedeficits in various forms of mild cognitive impairment, cognitivedeficits in various forms of dementia, dementia pugilistica, vascularand frontal lobe dementia, cognitive impairment, learning impairment,eye injuries, eye diseases, eye disorders, glaucoma, retinopathy,macular degeneration, head or brain or spinal cord injuries, head orbrain or spinal cord trauma, convulsions, epileptic convulsions,epilepsy, temporal lobe epilepsy, myoclonic epilepsy, tinnitus,dyskinesias, chorea, Huntington's chorea, athetosis, dystonia,stereotypy, ballism, tardive dyskinesias, tic disorder, torticollisspasmodicus, blepharospasm, focal and generalized dystonia, nystagmus,hereditary cerebellar ataxias, corticobasal degeneration, tremor,essential tremor, addiction, anxiety disorders, panic disorders, socialanxiety disorder (SAD), attention deficit hyperactivity disorder (ADHD),attention deficit syndrome (ADS), restless leg syndrome (RLS),hyperactivity in children, autism, dementia, dementia in Alzheimer'sdisease, dementia in Korsakoff syndrome, Korsakoff syndrome, vasculardementia, dementia related to HIV infections, HIV-1 encephalopathy, AIDSencephalopathy, AIDS dementia complex, AIDS-related dementia, majordepressive disorder, major depression, depression, memory loss, stress,bipolar manic-depressive disorder, drug tolerance, drug tolerance toopioids, movement disorders, fragile-X syndrome, irritable bowelsyndrome (IBS), migraine, multiple sclerosis (MS), muscle spasms, pain,chronic pain, acute pain, inflammatory pain, neuropathic pain,posttraumatic stress disorder (PTSD), schizophrenia, spasticity,Tourette's syndrome, eating disorders, food addiction, binge eatingdisorders, agoraphobia, generalized anxiety disorder,obsessive-compulsive disorder, panic disorder, social phobia, phobicdisorders, substance-induced anxiety disorder, delusional disorder,schizoaffective disorder, schizophreniform disorder, substance-inducedpsychotic disorder, hypertension, or any combination thereof.

The disease or disorder can be an expression-sensitive disease ordisorder. An expression-sensitive disease or disorder can becharacterized by decreased expression of one or more proteins, whereinectopic overexpression of said one or more proteins at a steady statelevel beyond the upper tuned threshold causes cellular toxicity and/ordisease. The disease or disorder can be a disease or disorder providedin Table 1. Table 1 provides an exemplary list of so-called “Goldilocks”diseases and disorders wherein disease phenotypes are attributable todecreased expression of genes but also exhibit cellular toxicity oroutright disease when overexpressed. The methods and compositionsprovided herein are surprisingly capable of treating or preventing saiddiseases and disorders via the tunable and robust expression meansprovided herein. In some embodiments, the payload gene comprises any ofthe genes provided in Table 1.

TABLE 1 EXPRESSION-SENSITIVE DISEASES AND DISORDERS Disorder ClassDisorder Gene Implicated in Disease Neurodevelopmental Rett SyndromeMeCP2 Syndromic Disorders Smith-Magenis Syndrome RAI1 Phelan-McDermidSyndrome SHANK3 Cornelia de Lange Syndrome and other NIPBL NIPBL relateddisorders DRK1A, KAT6A and related disorders of DRK1A, KAT6A severeintellectual disability Chromosome 2Q37 Deletion Syndrome and HDAC4other HDAC4 Related Disorders Angelman Syndrome UBE3A Kleefstra SyndromeEHMT1 and other genes encoded on chromosome 9q34.3 Joubert Syndrome andother NPHP1 Related NPHP1 Disorders Williams Syndrome LIMK1 and othergenes encoded on chromosome 7q11.23 Proliferative/CancerNeurofibromatosis Type 1 NF1 Disorders Li-Fraumeni syndrome and similarp53- P53 related cancer syndromes Glycogen Storage PhosphofructokinaseDeficiency PFK Disorders Hematologic/Immune X-linked Hyper IgM Syndromeand similar CD40L Disorders primary immunodeficiency disordersTriosephosphate isomerase deficiency TPI1 Endocrine Disorders KallmanSyndrome FGFR1 and related genes Aromatase Deficiency CYP19A1 OtherBatten Disease, Frontotemporal Dementia PGRN Neuropsychiatric and otherneurodegenerative disorders Disorders related to loss of progranulinCholinergic Receptor Nicotinic Alpha 7 CHRNA7 Subunit Related DisordersHereditary Neuropathy with liability to PMP22 Pressure PalsiesPromoters

The promoters of the nucleic acids provided herein can vary depending onthe embodiment. The promoter can comprise a ubiquitous promoter. Theubiquitous promoter can be selected from the group comprising acytomegalovirus (CMV) immediate early promoter, a CMV promoter, a viralsimian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemiavirus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, an RSVpromoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5,P7.5, and P11 promoters from vaccinia virus, an elongation factor1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H(FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase(GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heatshock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1(HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), thehuman ROSA 26 locus, a Ubiquitin C promoter (UBC), a phosphoglyceratekinase-1 (PGK) promoter, 3-phosphoglycerate kinase promoter, acytomegalovirus enhancer, human β-actin (HBA) promoter, chicken β-actin(CBA) promoter, a CAG promoter, a CBH promoter, or any combinationthereof.

In some embodiments, one or more cells of a subject (e.g., a human)comprise an endogenous version of the payload gene, and the promoter cancomprise or can be derived from the promoter of the endogenous version.The promoter can comprise at least about 25% (e.g., 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, or a number or a range between any two ofthese values) homology to the promoter of the endogenous version of thepayload gene. The promoter can be a methyl CpG binding protein 2 (MeCP2)promoter or a derivative thereof (e.g., a MeCP2 promoter truncated toabout 229 bp and/or a MeCP2 promoter truncated to about 406 bp). Thepromoter can comprise an intronic sequence. The promoter can comprise abidirectional promoter and/or an enhancer (e.g., a CMV enhancer).

The promoter can be an inducible promoter (e.g., a tetracyclineresponsive promoter, a TRE promoter, a Tre3G promoter, an ecdysoneresponsive promoter, a cumate responsive promoter, a glucocorticoidresponsive promoter, and estrogen responsive promoter, a PPAR-γpromoter, and/or an RU-486 responsive promoter). The promoter cancomprise a tissue-specific promoter and/or a lineage-specific promoter.The tissue specific promoter can be a liver-specific thyroxin bindingglobulin (TBG) promoter, an insulin promoter, a glucagon promoter, asomatostatin promoter, a pancreatic polypeptide (PPY) promoter, asynapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammaliandesmin (DES) promoter, a α-myosin heavy chain (a-MHC) promoter, or acardiac Troponin T (cTnT) promoter. The tissue specific promoter can bea neuron-specific promoter (e.g., a synapsin-1 (Syn) promoter, a CaMKIIapromoter, a calcium/calmodulin-dependent protein kinase II a promoter, atubulin alpha I promoter, a neuron-specific enolase promoter, aplatelet-derived growth factor beta chain promoter, TRPV1 promoter, aNa_(v)1.7 promoter, a Na_(v)1.8 promoter, a Na_(v)1.9 promoter, or anAdvillin promoter). The tissue specific promoter can be amuscle-specific promoter (e.g., a creatine kinase (MCK) promoter).

In some embodiments, payload expression can be gated by a drug/smallmolecule. In some embodiments, the method can comprise an induciblepromoter or a repressible promoter. The method can compriseadministering one or more doses (e.g., a higher starting dose and alower maintenance dose) of an agent that exerts an effect on thepromoter (e.g., an inducer of said inducible promoter). In some suchembodiments, only induce payload expression at a certain time point orexpression profile, such as, for example, cases where a starting higherdoes versus a longer term maintenance dose is needed. Some embodimentsof the compositions, methods, and systems provided herein can compriseone or more components of an transactivator rtTA (reversetetracycline-controlled transactivator) system. By using an rrTA system,expression of the gene of interest (e.g. payload) can be furtherregulated by an inducible system whereby only when a small moleculedoxycycline is added, the IFFL regulated construct is expressed.

The compositions provided herein can comprise a tetracycline-on (Tet-On)system. As an example, tetracycline-on (Tet-On) systems can use areverse tetracycline transactivator (rtTA) to induce gene expression.Reverse tetracycline transactivators (rtTAs) comprise a mutanttetracycline repressor DNA binding protein (TetR) and a transactivationdomain. These transactivators can be activated in the presence of atetracycline (e.g., doxycycline) and subsequently bind to promoterscomprising a tetracycline-responsive element (TRE) to induce geneexpression. A TRE comprises at least one Tet operator (Tet-O) sequence(e.g., multiple repeats of Tel-0 sequences) and may be located upstreamof a minimal promoter (e.g., minimal promoter sequence derived from thehuman cytomegalovirus (hCMV) immediate-early promoter). A “Tet-On”system, as used herein, is a type of inducible system that is capable ofinducing expression of a particular payload gene in the presence oftetracycline (e.g., doxycycline (DOX)). In certain embodiments, a Tet-Onsystem comprises a tetracycline-responsive promoter operably linked to apayload gene (e.g., a therapeutic sequence, a gene-targeting nucleicacid, and/or a nucleic acid encoding a protein) and a reversetetracycline-controlled transactivator (rtTA). The expression cassetteencoding a tetracycline-responsive promoter (e.g., a promoter comprisinga TRE, including TRE3G, P tight, and TRE2) and a reversetetracycline-controlled transactivator may be encoded on the same vectoror be encoded on separate vectors. In some embodiments, the promotercomprises Tet Response Element (TRE). Tetracycline-dependent promoterscan be constructed by placing a TRE upstream of a minimal promoter.

A “reverse tetracycline transactivator” (“rtTA”), as used herein, shallbe given its ordinary meaning, and shall also refer an inducing agentthat binds to a TRE promoter (e.g., a TRE3G, P tight, or TRE2 promoter)in the presence of tetracycline (e.g., doxycycline) and is capable ofdriving expression of a payload gene that is operably linked to the TREpromoter. rtTAs generally comprise a mutant tetracycline repressor DNAbinding protein (TetR) and a transactivation domain. Any suitabletransactivation domain may be used. Non-limiting examples include VP64,P65, RTA, and MPH MS2-P65-HSF1. In some embodiments, a rtTA of thepresent disclosure comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100transactivation domains. The mutant TetR domain is capable of binding toa TRE promoter when bound to tetracycline.

The methods and compositions provided herein can comprise a tetracyclinerepressor. The term “tetracycline repressor” or “TetR” shall be givenits ordinary meaning, and shall also refer to a protein that is capableof binding to a Tet-0 sequence (e.g., a Tet-0 sequence in a TRE) in theabsence of tetracycline (e.g., doxycycline) and prevents binding of rtTA(e.g., rtTA3, rtTA4, or variants thereof) in the absence of tetracycline(e.g., doxycycline). TetRs prevent gene expression from promoterscomprising a TRE in the absence of tetracycline (e.g., doxycycline). Inthe presence of tetracycline, TetRs cannot bind promoters comprising aTRE, and TetR cannot prevent transcription.

The term“promoter” shall be given its ordinary meaning, and shall alsorefer to a control region of a nucleic acid sequence at which initiationand rate of transcription of the remainder of a nucleic acid sequenceare controlled. A promoter may also contain sub-regions at whichregulatory proteins and molecules may bind, such as RNA polymerase andother transcription factors. Promoters may be constitutive, inducible,activatable, repressible, tissue-specific, or any combination thereof. Apromoter drives expression or drives transcription of the nucleic acidsequence that it regulates. Herein, a promoter is considered to be“operably linked” when it is in a correct functional location andorientation in relation to a nucleic acid sequence it regulates tocontrol (“drive”) transcriptional initiation of that sequence,expression of that sequence, or a combination thereof.

A promoter may promote ubiquitous expression or tissue-specificexpression of an operably linked nucleic acid sequence from any species,including humans. In some embodiments, the promoter is a eukaryoticpromoter. Non-limiting examples of eukaryotic promoters include TDH3,PGK1, PKC1, TDH2, PYK1, TPI1, AT1, CMV, EF1a, SV40, PGK1 (human ormouse), Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa,GAL1, GAL 10, TEF, GDS, ADH1, CaMV35S, Ubi, Hl, and U6, as would beknown to one of ordinary skill in the art.

Non-limiting examples of ubiquitous promoters includetetracycline-responsive promoters (under the relevant conditions), CMV,EF1 alpha, a SV40 promoter, PGK1, Ubc, CAG, human beta actin genepromoter, and a promoter comprising an upstream activating sequence(UAS). In certain embodiments, the promoter is a mammalian promoter.Non-limiting examples of tissue-specific promoters includebrain-specific, liver-specific, muscle-specific, nerve cell-specific,lung-specific, heart-specific, bone-specific, intestine-specific,skin-specific promoters, brain-specific promoters, and eye-specificpromoters.

Non-limiting examples of constitutive promoters include CP1, CMV, EF1alpha, SV40, PGK1, Ubc, human beta actin, beta tubulin, CAG, Ac5,polyhedrin, TEF1, GDS, CaM3 5S, Ubi, Hl, and U6.

An “inducible promoter” shall be given its ordinary meaning, and shallalso refer one that is characterized by initiating or enhancingtranscriptional activity when in the presence of, influenced by, orcontacted by an inducing agent. An inducing agent may be endogenous or anormally exogenous condition, compound, agent, or protein that contactsan engineered nucleic acid in such a way as to be active in inducingtranscriptional activity from the inducible promoter. In certainembodiments, an inducing agent is a tetracycline-sensitive protein(e.g., rtTA).

Inducible promoters for use in accordance with the present disclosureinclude any inducible promoter described herein or known to one ofordinary skill in the art. Examples of inducible promoters include,without limitation, chemically/biochemically-regulated andphysically-regulated promoters such as alcohol-regulated promoters,tetracycline-regulated promoters (e.g., anhydrotetracycline(aTc)-responsive promoters and other tetracycline responsive promotersystems, which include a tetracycline repressor protein (tetRTetR), atetracycline operator sequence (tetO), and a tetracycline transactivatorfusion protein (tTA), and a tetracycline operator sequence (tetO) and areverse tetracycline transactivator fusion protein (rtTA)),steroid-regulated promoters (e.g., promoters based on the ratglucocorticoid receptor, human estrogen receptor, moth ecdysonereceptors, and promoters from the steroid/retinoid/thyroid 25 receptorsuperfamily), metal-regulated promoters (e.g., promoters derived frommetallothionein (proteins that bind and sequester metal ions) genes fromyeast, mouse and human), pathogenesis-regulated promoters (e.g., inducedby salicylic acid, ethylene or benzothiadiazole (BTH)),temperature/heat-inducible promoters (e.g., heat shock promoters), andlight-regulated promoters.

Payloads

In some embodiments, the payload protein comprises a disease-associatedprotein, wherein aberrant expression of the disease-associated proteincorrelates with the occurrence and/or progression of the disease. Thepayload protein can comprise a protein associated with anexpression-sensitive disease or disorder as provided in Table 1. Thepayload protein can comprise methyl CpG binding protein 2 (MeCP2),DRK1A, KAT6A, NIPBL, HDAC4, UBE3A, EHMT1, one or more genes encoded onchromosome 9q34.3, NPHP1, LIMK1 one or more genes encoded on chromosome7q11.23, P53, TPI1, FGFR1 and related genes, RA1, SHANK3, CLN3, NF-1,TP53, PFK, CD40L, CYP19A1, PGRN, CHRNA7, PMP22, CD40LG, derivativesthereof, or any combination thereof.

The payload protein can comprise fluorescence activity, polymeraseactivity, protease activity, phosphatase activity, kinase activity,SUMOylating activity, deSUMOylating activity, ribosylation activity,deribosylation activity, myristoylation activity demyristoylationactivity, or any combination thereof.

The payload protein can comprise nuclease activity, methyltransferaseactivity, demethylase activity, DNA repair activity, DNA damageactivity, deamination activity, dismutase activity, alkylation activity,depurination activity, oxidation activity, pyrimidine dimer formingactivity, integrase activity, transposase activity, recombinaseactivity, polymerase activity, ligase activity, helicase activity,photolyase activity, glycosylase activity, acetyltransferase activity,deacetylase activity, adenylation activity, deadenylation activity, orany combination thereof.

The payload protein can comprise a programmable nuclease. Theprogrammable nuclease can be selected from the group comprising: SpCas9or a derivative thereof, VRER, VQR, EQR SpCas9; xCas9-3.7; eSpCas9;Cas9-HF1; HypaCas9; evoCas9; HiFi Cas9; ScCas9; StCas9; NmCas9; SaCas9;CjCas9; CasX; Cas9 H940A nickase; Cas12 and derivatives thereof,dcas9-APOBEC1 fusion, BE3, and dcas9-deaminase fusions; dcas9-Krab,dCas9-VP64, dCas9-Tet1, and dcas9-transcriptional regulator fusions;Dcas9-fluorescent protein fusions; Cas13-fluorescent protein fusions;RCas9-fluorescent protein fusions; Cas13-adenosine deaminase fusions.The programmable nuclease can comprise a zinc finger nuclease (ZFN)and/or transcription activator-like effector nuclease (TALEN). Theprogrammable nuclease can comprise Streptococcus pyogenes Cas9 (SpCas9),Staphylococcus aureus Cas9 (SaCas9), a zinc finger nuclease, TALeffector nuclease, meganuclease, MegaTAL, Tev-m TALEN, MegaTev, homingendonuclease, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8,Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e,Cas13a, Cas13b, Cas13c, derivatives thereof, or any combination thereof.In some embodiments, the nucleic acid further comprises a polynucleotideencoding (i) a targeting molecule and/or (ii) a donor nucleic acid. Thecomposition can comprise (i) a targeting molecule or a nucleic acidencoding the targeting molecule and/or (ii) a donor nucleic acid or anucleic acid encoding the donor nucleic acid. The targeting molecule canbe capable of associating with the programmable nuclease. The targetingmolecule can comprise single strand DNA or single strand RNA. Thetargeting molecule can comprise a single guide RNA (sgRNA). Theprogrammable nuclease can comprise a zinc finger nuclease, TAL effectornuclease, meganuclease, MegaTAL, Tev-m TALEN, MegaTev, homingendonuclease, derivatives thereof, or any combination thereof. Thetargeting molecule can be capable of associating with the programmablenuclease. The targeting molecule can comprise single strand DNA orsingle strand RNA. The targeting molecule can comprise a single guideRNA (sgRNA). The targeting molecule can comprise a synthetic nucleicacid.

In some embodiments, the payload comprises one or more programmablenucleases disclosed in Table 2. In some embodiments, the payloadcomprises one or more prime editors. There are provided, in someembodiments, methods of gene editing that fulfill the gene purposeand/or regulation purpose set forth in Table 2.

TABLE 2 PROGRAMMABLE NUCLEASES Gene Gene Purpose Regulation PurposeSpCas9 Genome Engineering, Reduce off-target nuclease activity or geneediting immunogenicity mutations thereof: VRER, VQR, and EQR GenomeEngineering, Reduce off-target nuclease activity or SpCas9 gene editingimmunogenicity xCas9-3.7 eSpCas9 Cas9-HF1 HypaCas9 evoCas9 HiFi Cas9Other Cas9 species: ScCas9 Genome Engineering, Reduce off-targetnuclease activity or StCas9 gene editing immunogenicity NmCas9 SaCas9CjCas9 CasX Cas9 H940A nickase Prime editing Reduce off-target editingand immunogenicity. Cas12 and mutations Multiplex gene editing Reduceoff-target nuclease activity or immunogenicity dcas9-APOBEC1 fusion,BE3, CRISPR base editing Reduce off-target base editing (which otherdcas9-deaminase fusions is a significant unsolved problem) orimmunogenicity dcas9-Krab, dCas9-VP64, activate/repress Reduceoff-target, decrease dCas9-Tet1, and other dcas9- transcription, modifyimmunogenicity. transcriptional regulator fusion epigenetic stateDcas9-fluorescent protein Imaging and tracking Increase signal to noiseratio fusions genomic loci and chromatin dynamics Cas13-fluorescentprotein RNA imaging and Increase signal to noise ratio fusions trackingRCas9-fluorescent protein RNA imaging Increase signal to noise ratiofusions Cas13-adenosine deaminase RNA editing reduce off-target andimmunogenicity fusions

The payload protein can comprise a chimeric antigen receptor. Themethods and compositions provided herein find use in cell therapies(e.g., adoptive therapies). Aspects of the invention accordingly involvethe adoptive transfer of immune system cells, such as T cells, specificfor selected antigens, such as tumor associated antigens. Variousstrategies may, for example, be employed to genetically modify T cellsby altering the specificity of the T cell receptor (TCR) for example byintroducing payloads comprising new TCR a and b chains with selectedpeptide specificity. As an alternative to, or addition to, TCRmodifications, chimeric antigen receptors (CARs) may be used in order togenerate immunoresponsive cells, such as T cells, specific for selectedtargets, such as malignant cells, with a wide variety of receptorchimera constructs having been described. In some embodiments, theinvention described herein relates to a method for adoptiveimmunotherapy, comprising (1) knock-in an exogenous gene encoding achimeric antigen receptor (CAR) or a T-cell receptor (TCR), (2)knock-out or knock-down expression of an immune checkpoint receptor, (3)knock-out or knock-down expression of an endogenous TCR, (4) knock-outor knock-down expression of a human leukocyte antigen class I (HLA-I)proteins, and/or (5) knock-out or knock-down expression of an endogenousgene encoding an antigen targeted by an exogenous CAR or TCR.

The payload protein can be associated with an agricultural trait ofinterest selected from the group consisting of increased yield,increased abiotic stress tolerance, increased drought tolerance,increased flood tolerance, increased heat tolerance, increased cold andfrost tolerance, increased salt tolerance, increased heavy metaltolerance, increased low-nitrogen tolerance, increased diseaseresistance, increased pest resistance, increased herbicide resistance,increased biomass production, male sterility, or any combinationthereof.

The payload protein can be associated with a biological manufacturingprocess selected from the group comprising fermentation, distillation,biofuel production, production of a compound, production of apolypeptide, or any combination thereof.

In some embodiments, the polynucleotide further encodes one or moresecondary proteins (e.g., secondary payload proteins). The secondarypayload proteins can comprise any of the payloads described herein. Thepayload protein and the one or more secondary proteins can be expressedas a fusion protein (and can be separated by one or more self-cleavingpeptides). The 3′UTR of the transgene(s) encoding the one or moresecondary proteins can comprise one or more silencer effector bindingsequences. The payload protein and the one or more secondary proteinscan be expressed on separate payload transcripts.

Protein-level circuits that can be expressed on a single transcript area growing trend in bioengineering. The behavior of protein-levelcircuits will depend on the expression levels of their individualcomponents. IFFLs can help guarantee correct functioning of thesecircuits by maintaining the stoichiometric ratios of circuit componentsin variable environments, including by regulating the circuit as a wholeif it is on a single transcript.

The payload protein and the one or more secondary proteins can beencoded on a single transcript, and wherein translations of the payloadprotein and the one or more secondary proteins can be each driven by aseparate internal ribosome entry site. The sequences of the internalribosome entry sites can be identical or different.

The payload protein and the one or more secondary proteins can comprisea synthetic protein circuit. Synthetic biology allows for rationaldesign of circuits that confer new functions in living cells. Manynatural cellular functions are implemented by protein-level circuits, inwhich proteins specifically modify each other's activity, localization,or stability. Synthetic protein circuits have been described in, Gao,Xiaojing J., et al. “Programmable protein circuits in living cells.”Science 361.6408 (2018): 1252-1258; and PCT Application Publication No.WO 2019/147478; the content of each of these, including any supportingor supplemental information or material, is incorporated herein byreference in its entirety. The methods and compositions provided hereincan express, for example, synthetic protein circuits that respond toinputs only above or below a certain tunable threshold concentration,such as those provided in U.S. patent application Ser. No. 16/738,664,published as US Patent Publication No. 2020/0277333, the content ofwhich is incorporated herein by reference in its entirety. The methodsand compositions provided herein can express, for example, syntheticprotein circuits comprising one or more synthetic protein circuit designcomponents and/or concepts of U.S. application Ser. No. 16/556,063,filed on Aug. 29, 2019, the content of which is incorporated herein byreference in its entirety.

Disclosed herein are nucleic acids comprising a polynucleotide encodingone or more payload genes. As disclosed herein, the payload gene isoperatively linked with appropriate regulatory elements in someembodiments. The one or more payload genes of the nucleic acid cancomprise a siRNA, a shRNA, an antisense RNA oligonucleotide, anantisense miRNA, a trans-splicing RNA, a guide RNA, single-guide RNA,crRNA, a tracrRNA, a trans-splicing RNA, a pre-mRNA, a mRNA, or anycombination thereof. The one or more payload genes of the nucleic acidcan comprise one or more synthetic protein circuit components. The oneor more payload genes of the nucleic acid can comprise can entiresynthetic protein circuit comprising one or more synthetic proteincircuit components. The one or more payload genes of the nucleic acidcan comprise two or more synthetic protein circuits.

The payload protein can be any protein, including naturally-occurringand non-naturally occurring proteins. Examples of payload proteininclude, but are not limited to, luciferases; fluorescent proteins(e.g., GFP); growth hormones (GHs) and variants thereof; insulin-likegrowth factors (IGFs) and variants thereof; granulocytecolony-stimulating factors (G-CSFs) and variants thereof, erythropoietin(EPO) and variants thereof, insulin, such as proinsulin, preproinsulin,insulin, insulin analogs, and the like; antibodies and variants thereof,such as hybrid antibodies, chimeric antibodies, humanized antibodies,monoclonal antibodies; antigen binding fragments of an antibody (Fabfragments), single-chain variable fragments of an antibody (scFVfragments); dystrophin and variants thereof, clotting factors andvariants thereof, cystic fibrosis transmembrane conductance regulator(CFTR) and variants thereof; and interferons and variants thereof.

In some embodiments, the payload protein is a therapeutic protein orvariant thereof. Non-limiting examples of therapeutic proteins includeblood factors, such as β-globin, hemoglobin, tissue plasminogenactivator, and coagulation factors; colony stimulating factors (CSF);interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, etc.; growth factors, such as keratinocyte growth factor (KGF),stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGFand acidic FGF), hepatocyte growth factor (HGF), insulin-like growthfactors (IGFs), bone morphogenetic protein (BMP), epidermal growthfactor (EGF), growth differentiation factor-9 (GDF-9), hepatoma derivedgrowth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF),neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin(TPO), transforming growth factor alpha (TGF-a), transforming growthfactor beta (TGF-β), and the like; soluble receptors, such as solubleTNF-receptors, soluble VEGF receptors, soluble interleukin receptors(e.g., soluble IL-1 receptors and soluble type II IL-1 receptors),soluble γ/δ T cell receptors, ligand-binding fragments of a solublereceptor, and the like; enzymes, such as β-glucosidase, imiglucarase,β-glucocerebrosidase, and alglucerase; enzyme activators, such as tissueplasminogen activator; chemokines, such as IP-10, monokine induced byinterferon-gamma (Mig), Gro/IL-8, RANTES, MIP-1, MIP-I β, MCP-1, PF-4,and the like; angiogenic agents, such as vascular endothelial growthfactors (VEGFs, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), transforminggrowth factor-beta, basic fibroblast growth factor, glioma-derivedgrowth factor, angiogenin, angiogenin-2; and the like; anti-angiogenicagents, such as a soluble VEGF receptor; protein vaccine; neuroactivepeptides, such as nerve growth factor (NGF), bradykinin,cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasinghormone, beta-endorphin, enkephalin, substance P, somatostatin,prolactin, galanin, growth hormone-releasing hormone, bombesin,dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y,luteinizing hormone, calcitonin, insulin, glucagons, vasopressin,angiotensin II, thyrotropin-releasing hormone, vasoactive intestinalpeptide, a sleep peptide, and the like; thrombolytic agents; atrialnatriuretic peptide; relaxin; glial fibrillary acidic protein; folliclestimulating hormone (FSH); human alpha-1 antitrypsin; leukemiainhibitory factor (LIF); transforming growth factors (TGFs); tissuefactors, luteinizing hormone; macrophage activating factors; tumornecrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growthfactor; tissue inhibitors of metalloproteinases; vasoactive intestinalpeptide; angiogenin; angiotropin; fibrin; hirudin; IL-1 receptorantagonists; and the like. Some other non-limiting examples of payloadprotein include ciliary neurotrophic factor (CNTF); brain-derivedneurotrophic factor (BDNF); neurotrophins 3 and 4/5 (NT-3 and 4/5);glial cell derived neurotrophic factor (GDNF); aromatic amino aciddecarboxylase (AADC); hemophilia related clotting proteins, such asFactor VIII, Factor IX, Factor X; dystrophin or mini-dystrophin;lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storagedisease-related enzymes, such as glucose-6-phosphatase, acid maltase,glycogen debranching enzyme, muscle glycogen phosphorylase, liverglycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase(e.g., PHKA2), glucose transporter (e.g., GLUT2), aldolase A, β-enolase,and glycogen synthase; lysosomal enzymes (e.g.,beta-N-acetylhexosaminidase A); and any variants thereof.

In some embodiments, the payload protein is an active fragment of aprotein, such as any of the aforementioned proteins. In someembodiments, the payload protein is a fusion protein comprising some orall of two or more proteins. In some embodiments a fusion protein cancomprise all or a portion of any of the aforementioned proteins.

In some embodiments, the payload protein is a multi-subunit protein. Forexamples, the payload protein can comprise two or more subunits, or twoor more independent polypeptide chains. In some embodiments, the payloadprotein can be an antibody. Examples of antibodies include, but are notlimited to, antibodies of various isotypes (for example, IgG1, IgG2,IgG3, IgG4, IgA, IgD, IgE, and IgM); monoclonal antibodies produced byany means known to those skilled in the art, including anantigen-binding fragment of a monoclonal antibody; humanized antibodies;chimeric antibodies; single-chain antibodies; antibody fragments such asFv, F(ab′)2, Fab′, Fab, Facb, scFv and the like; provided that theantibody is capable of binding to antigen. In some embodiments, theantibody is a full-length antibody.

In some embodiments, the payload gene encodes a pro-survival protein(e.g., Bcl-2, Bcl-XL, Mcl-1 and A1). In some embodiments, the payloadgene encodes a apoptotic factor or apoptosis-related protein such as,for example, AIF, Apaf (e.g., Apaf-1, Apaf-2, and Apaf-3), oder APO-2(L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl-xL, Bcl-xs, bik, CAD,Calpain, Caspase (e.g., Caspase-1, Caspase-2, Caspase-3, Caspase-4,Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, andCaspase-11), ced-3, ced-9, c-Jun, c-Myc, crm A, cytochrom C, CdR1, DcR1,DD, DED, DISC, DNA-PKcs, DR3, DR4, DR5, FADD/MORT-1, FAK, Fas(Fas-ligand CD95/fas (receptor)), FLICE/MACH, FLIP, fodrin, fos,G-Actin, Gas-2, gelsolin, granzyme A/B, ICAD, ICE, INK, Lamin A/B, MAP,MCL-1, Mdm-2, MEKK-1, MORT-1, NEDD, NF-_(kappa)B, NuMa, p53, PAK-2,PARP, perforin, PITSLRE, PKCdelta, pRb, presenilin, prICE, RAIDD, Ras,RIP, sphingomyelinase, thymidinkinase from herpes simplex, TRADD, TRAF2,TRAIL-R1, TRAIL-R2, TRAIL-R3, and/or transglutaminase.

In some embodiments, the payload gene encodes a cellular reprogrammingfactor capable of converting an at least partially differentiated cellto a less differentiated cell, such as, for example, Oct-3, Oct-4, Sox2,c-Myc, Klf4, Nanog, Lin28, ASCL1, MYT1 L, TBX3b, SV40 large T, hTERT,miR-291, miR-294, miR-295, or any combinations thereof. In someembodiments, the payload gene encodes a programming factor that iscapable of differentiating a given cell into a desired differentiatedstate, such as, for example, nerve growth factor (NGF), fibroblastgrowth factor (FGF), interleukin-6 (IL-6), bone morphogenic protein(BMP), neurogenin3 (Ngn3), pancreatic and duodenal homeobox 1 (Pdx1),Mafa, or any combination thereof.

In some embodiments, the payload gene encodes a human adjuvant proteincapable of eliciting an innate immune response, such as, for example,cytokines which induce or enhance an innate immune response, includingIL-2, IL-12, IL-15, IL-18, IL-21CCL21, GM-CSF and TNF-alpha; cytokineswhich are released from macrophages, including IL-1, IL-6, IL-8, IL-12and TNF-alpha; from components of the complement system including C1q,MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b,C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4 bp, MCP, DAF, H, I,P and CD59; from proteins which are components of the signaling networksof the pattern recognition receptors including TLR and IL-1 R1, whereasthe components are ligands of the pattern recognition receptorsincluding IL-1 alpha, IL-1 beta, Beta-defensin, heat shock proteins,such as HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen,Typlll repeat extra domain A of fibronectin; the receptors, includingIL-1 RI, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10,TLR11; the signal transducers including components of the Small-GTPasessignaling (RhoA, Ras, Rac1, Cdc42 etc.), components of the PIP signaling(PI3K, Src-Kinases, etc.), components of the MyD88-dependent signaling(MyD88, IRAK1, IRAK2, etc.), components of the MyD88-independentsignaling (TICAM1, TICAM2 etc.); activated transcription factorsincluding e.g. NF-κB, c-Fos, c-Jun, c-Myc; and induced target genesincluding e.g. IL-1 alpha, IL-1 beta, Beta-Defensin, IL-6, IFN gamma,IFN alpha and IFN beta; from costimulatory molecules, including CD28 orCD40-ligand or PD1; protein domains, including LAMP; cell surfaceproteins; or human adjuvant proteins including CD80, CD81, CD86, trif,flt-3 ligand, thymopentin, Gp96 or fibronectin, etc., or any specieshomolog of any of the above human adjuvant proteins.

In some embodiments, the payload gene encodes immunogenic materialcapable of stimulating an immune response (e.g., an adaptive immuneresponse) such as, for example, antigenic peptides or proteins from apathogen. The expression of the antigen may stimulate the body'sadaptive immune system to provide an adaptive immune response. Thus, itis contemplated that some embodiments the nucleic acids provided hereincan be employed as vaccines for the prophylaxis or treatment ofinfectious diseases (e.g., as vaccines).

As described herein, the nucleotide sequence encoding the payloadprotein can be modified to improve expression efficiency of the protein.The methods that can be used to improve the transcription and/ortranslation of a gene herein are not particularly limited. For example,the nucleotide sequence can be modified to better reflect host codonusage to increase gene expression (e.g., protein production) in the host(e.g., a mammal).

The degree of payload gene expression in the target cell can vary. Forexample, in some embodiments, the payload gene encodes a payloadprotein. The amount of the payload protein expressed in the subject(e.g., the serum of the subject) can vary. For example, in someembodiments the protein can be expressed in the serum of the subject inthe amount of at least about 9 μg/ml, at least about 10 μg/ml, at leastabout 50 μg/ml, at least about 100 μg/ml, at least about 200 g/ml, atleast about 300 μg/ml, at least about 400 μg/ml, at least about 500μg/ml, at least about 600 μg/ml, at least about 700 μg/ml, at leastabout 800 μg/ml, at least about 900 μg/ml, or at least about 1000 μg/ml.In some embodiments, the payload protein is expressed in the serum ofthe subject in the amount of about 9 g/ml, about 10 μg/ml, about 50μg/ml, about 100 μg/ml, about 200 μg/ml, about 300 μg/ml, about 400μg/ml, about 500 μg/ml, about 600 μg/ml, about 700 μg/ml, about 800μg/ml, about 900 μg/ml, about 1000 μg/ml, about 1500 μg/ml, about 2000μg/ml, about 2500 μg/ml, or a range between any two of these values. Askilled artisan will understand that the expression level in which apayload protein is needed for the method to be effective can varydepending on non-limiting factors such as the particular payload proteinand the subject receiving the treatment, and an effective amount of theprotein can be readily determined by a skilled artisan usingconventional methods known in the art without undue experimentation.

A payload protein encoded by a payload gene can be of various lengths.For example, the payload protein can be at least about 200 amino acids,at least about 250 amino acids, at least about 300 amino acids, at leastabout 350 amino acids, at least about 400 amino acids, at least about450 amino acids, at least about 500 amino acids, at least about 550amino acids, at least about 600 amino acids, at least about 650 aminoacids, at least about 700 amino acids, at least about 750 amino acids,at least about 800 amino acids, or longer in length. In someembodiments, the payload protein is at least about 480 amino acids inlength. In some embodiments, the payload protein is at least about 500amino acids in length. In some embodiments, the payload protein is about750 amino acids in length.

The payload genes can have different lengths in differentimplementations. The number of payload genes can be different indifferent embodiments. In some embodiments, the number of payload genesin a nucleic acid can be, or can be about, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or anumber or a range between any two of these values. In some embodiments,the number of payload genes in a nucleic acid can be at least, or can beat most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25. In some embodiments, a payload genes is, oris about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250,3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500,8000, 8500, 9000, 9500, 10000, or a number or a range between any two ofthese values, nucleotides in length. In some embodiments, a payload geneis at least, or is at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790,800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930,940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800,2900, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000,6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10000 nucleotides inlength.

The payload can be an inducer of cell death. The payload can be inducecell death by a non-endogenous cell death pathway (e.g., a bacterialpore-forming toxin). In some embodiments, the payload can be apro-survival protein. In some embodiments, the payload is a modulator ofthe immune system. The payload can activate an adaptive immune response,and innate immune response, or both. In some embodiments, the payloadgene encodes immunogenic material capable of stimulating an immuneresponse (e.g., an adaptive immune response) such as, for example,antigenic peptides or proteins from a pathogen. The expression of theantigen may stimulate the body's adaptive immune system to provide anadaptive immune response. Thus, it is contemplated that some embodimentsthe compositions provided herein can be employed as vaccines for theprophylaxis or treatment of infectious diseases (e.g., as vaccines). Thepayload protein can comprise a CRE recombinase, GCaMP, a cell therapycomponent, a knock-down gene therapy component, a cell-surface exposedepitope, or any combination thereof.

Examples of payload genes include a sequence associated with a signalingbiochemical pathway, e.g., a signaling biochemical pathway-associatedgene or polynucleotide (e.g., a signal transducer). In some embodiments,the methods and compositions disclosed herein comprise knockdown of anendogenous signal transducer accompanied by tuned expression of apayload protein comprising an appropriate version of signal transducer.Examples of target polynucleotides include a disease associated gene orpolynucleotide. A “disease-associated” gene or polynucleotide refers toany gene or polynucleotide which is yielding transcription ortranslation products at an abnormal level or in an abnormal form incells derived from a disease-affected tissues compared with tissues orcells of a non-disease control. It may be a gene that becomes expressedat an abnormally high level; it may be a gene that becomes expressed atan abnormally low level, where the altered expression correlates withthe occurrence and/or progression of the disease. A disease-associatedgene also refers to a gene possessing mutation(s) or genetic variationthat is directly responsible or is in linkage disequilibrium with agene(s) that is responsible for the etiology of a disease. Thetranscribed or translated products may be known or unknown, and may beat a normal or abnormal level. Signal transducers can be can beassociated with one or more diseases or disorders. In some embodiments,a disease or disorder is characterized by an aberrant signaling of oneor more signal transducers disclosed herein. In some embodiments, theactivation level of the signal transducer correlates with the occurrenceand/or progression of a disease or disorder. The activation level of thesignal transducer can be directly responsible or indirectly responsiblefor the etiology of the disease or disorder. Non-limiting examples ofsignal transducers, signal transduction pathways, and diseases anddisorders characterized by aberrant signaling of said signal transducersare listed in Tables 3-5. In some embodiments, the methods andcompositions disclosed herein prevent or treat one or more of thediseases and disorders listed in Tables 3-5. In some embodiments, thepayload comprises a replacement version of the signal transducer. Insome embodiments, the methods and compositions further compriseknockdown of the corresponding endogenous signal transducer. The payloadcan comprise the product of a gene listed in listed in Tables 3-5. Insome embodiments, the payload ameliorates a disease or disordercharacterized by an aberrant signaling of one or more signalingtransducers. In some embodiments, the payload diminishes the activationlevel of one or more signal transducers (e.g., signal transducers withaberrant overactive signaling, signal transducers listed in Tables 3-5).In some embodiments, the payload increases the activation level of oneor more signal transducers (e.g., signal transducers with aberrantunderactive signaling). In some such embodiments, the payload canmodulate the abundance, location, stability, and/or activity ofactivators or repressors of said signal transducers.

TABLE 3 DISEASES AND DISORDERS OF INTEREST Diseases/Disorders GenesNeoplasia PTEN; ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4; Notch1; Notch2;Notch3; Notch4; AKT; AKT2; AKT3; HIF; HIF1a; HIF3a; Met; HRG; Bcl2; PPARalpha; PPAR gamma; WT1 (Wilms Tumor); FGF Receptor Family members (5members: 1, 2, 3, 4, 5); CDKN2a; APC; RB (retinoblastoma); MEN1; VHL;BRCA1; BRCA2; AR (Androgen Receptor); TSG101; IGF; IGF Receptor; Igf1 (4variants); Igf2 (3 variants); Igf 1 Receptor; Igf 2 Receptor; Bax; Bcl2;caspases family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; ApcAge-related Macular Abcr; Ccl2; Cc2; cp (ceruloplasmin); Timp3;cathepsinD; Vldlr; Ccr2 Degeneration Schizophrenia Neuregulin1 (Nrg1);Erb4 (receptor for Neuregulin); Complexin1 (Cplx1); Tph1 Tryptophanhydroxylase; Tph2 Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a;GSK3b Disorders 5-HTT (Slc6a4); COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1;Dao (Dao1) Trinucleotide Repeat HTT (Huntington's Dx); SBMA/SMAX1/AR(Kennedy's Dx); FXN/X25 Disorders (Friedrich's Ataxia); ATX3 (Machado-Joseph's Dx); ATXN1 and ATXN2 (spinocerebellar ataxias); DMPK (myotonicdystrophy); Atrophin-1 and Atn1 (DRPLA Dx); CBP (Creb-BP - globalinstability); VLDLR (Alzheimer's); Atxn7; Atxn10 Fragile X SyndromeFMR2; FXR1; FXR2; mGLUR5 Secretase Related APH-1 (alpha and beta);Presenilin (Psen1); nicastrin (Ncstn); PEN-2 Disorders Others Nos1;Parp1; Nat1; Nat2 Prion-related disorders Prp ALS SOD1; ALS2; STEX; FUS;TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c) Drug addiction Prkce (alcohol);Drd2; Drd4; ABAT (alcohol); GRIA2; Grm5; Grin1; Htr1b; Grin2a; Drd3;Pdyn; Gria1 (alcohol) Autism Mecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1;Fragile X (FMR2 (AFF2); FXR1; FXR2; Mglur5) Alzheimer's Disease E1;CHIP; UCH; UBB; Tau; LRP; PICALM; Clusterin; PS1; SORL1; CR1; Vldlr;Uba1; Uba3; CHIP28 (Aqp1, Aquaporin 1); Uchl1; Uchl3; APP InflammationIL-10; IL-1 (IL-1a; IL-1b); IL-13; IL-17 (IL-17a (CTLA8); IL- 17b;IL-17c; IL- 17d; IL-17f); II-23; Cx3cr1; ptpn22; TNFa; NOD2/CARD15 forIBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4; Cx3cl1 Parkinson's Diseasex-Synuclein; DJ-1; LRRK2; Parkin; PINK1

TABLE 4 SIGNAL TRANSDUCERS Blood and Anemia (CDAN1, CDA1, RPS19, DBA,PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, coagulation RH50A, NRAMP2, SPTB,ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT); Bare diseases and lymphocytesyndrome (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, disordersC2TA, RFX5, RFXAP, RFX5); Bleeding disorders (TBXA2R, P2RX1, P2X1);Factor H and factor H-like 1 (HF1, CFH, HUS); Factor V and factor VIII(MCFD2); Factor VII deficiency (F7); Factor X deficiency (F10); FactorXI deficiency (F11); Factor XII deficiency (F12, HAF); Factor XIIIAdeficiency (F13A1, F13A); Factor XIIIB deficiency (F13B); Fanconi anemia(FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FLJ34064, FANCB, FANCC,FACC, BRCA2, FANCD1, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF,XRCC9, FANCG, BRIP1, BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596);Hemophagocytic lymphohistiocytosis disorders (PRF1, HPLH2, UNC13D,MUNC13-4, HPLH3, HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B(F9, HEMB), Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficienciesand disorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3,EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia(HBA2, HBB, HBD, LCRB, HBA1). Cell B-cell non-Hodgkin lymphoma (BCL7A,BCL7); Leukemia (TAL1 TCL5, SCL, TAL2, dysregulation FLT3, NBS1, NBS,ZNFN1A1, IK1, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, and oncology ARNT,KRAS2, RASK2, GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM, diseases andCLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214,D9S46E, disorders CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3, FLT3,AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM, CLTH,ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS,NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1,GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN).Inflammation AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12,SDF1); and immune Autoimmune lymphoproliferative syndrome (TNFRSF6,APT1, FAS, CD95, related ALPS1A); Combined immunodeficiency, (IL2RG,SCIDX1, SCIDX, IMD4); HIV-1 diseases and (CCL5, SCYA5, D17S136E,TCP228), HIV susceptibility or infection (IL10, CSIF, disorders CMKBR2,CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G, AICDA,AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM,FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI); Inflammation (IL-10,IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c,IL-17d, IL- 17f), 11-23, Cx3cr1, ptpn22, TNFa, NOD2/CARD15 for IBD,IL-6, IL-12 (IL-12a, IL- 12b), CTLA4, Cx3cl1); Severe combinedimmunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1,RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX,IMD4). Metabolic, Amyloid neuropathy (TTR, PALB); Amyloidosis (APOA1,APP, AAA, CVAP, AD1, liver, kidney GSN, FGA, LYZ, TTR, PALB); Cirrhosis(KRT18, KRT8, CIRH1A, NAIC, TEX292, and protein KIAA1988); Cysticfibrosis (CFTR, ABCC7, CF, MRP7); Glycogen storage diseases diseases and(SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1,disorders GYS2, PYGL, PFKM); Hepatic adenoma (TCF1, HNF1A, MODY3),Hepatic failure, early onset, and neurologic disorder (SCOD1, SCO1),Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancer and carcinomas(CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1,IGF2R, MPRI, MET, CASP8, MCH5); Medullary cystic kidney disease (UMOD,HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1, QDPR, DHPR,PTS); Polycystic kidney and hepatic disease (FCYT, PKHD1, ARPKD, PKD1,PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63). Muscular/ Beckermuscular dystrophy (DMD, BMD, MYF6), Duchenne Muscular DystrophySkeletal (DMD, BMD); Emery-Dreifuss muscular dystrophy (LMNA, LMN1,EMD2, FPLD, diseases and CMD1A, HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD,CMD1A); disorders Facioscapulohumeral muscular dystrophy (FSHMD1A,FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE,KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG,LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD,SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP,MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1,SELN, RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1, LRP7, LR3,OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7, OC116,OPTB1); Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3,SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1,SMARD1). Neurological ALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a,VEGF-b, VEGF-c); and neuronal Alzheimer disease (APP, AAA, CVAP, AD1,APOE, AD2, PSEN2, AD4, STM2, diseases and APBB2, FE65L1, NOS3, PLAU,URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, disorders PTIP, A2M, BLMH,BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1,GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2);Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5); Huntington's disease anddisease like disorders (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP,SCA17); Parkinson disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17,SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1,PARK5, SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2); Rettsyndrome (MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX,MRX16, MRX79, x-Synuclein, DJ-1); Schizophrenia (Neuregulin1 (Nrg1),Erb4 (receptor for Neuregulin), Complexin1 (Cplx1), Tph1 Tryptophanhydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a,GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd1a), SLC6A3, DAOA, DTNBP1, Dao(Dao1)); Secretase Related Disorders (APH-1 (alpha and beta), Presenilin(Psen1), nicastrin, (Ncstn), PEN-2, Nos1, Parp1, Nat1, Nat2);Trinucleotide Repeat Disorders (HTT (Huntington's Dx), SBMA/SMAX1/AR(Kennedy's Dx), FXN/X25 (Friedrich's Ataxia), ATX3 (Machado- Joseph'sDx), ATXN1 and ATXN2 (spinocerebellar ataxias), DMPK (myotonicdystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP (Creb-BP - globalinstability), VLDLR (Alzheimer's), Atxn7, Atxn10). Ocular Age-relatedmacular degeneration (Abcr, Ccl2, Cc2, cp (ceruloplasmin), Timp3,diseases and cathepsinD, Vldlr, Ccr2); Cataract (CRYAA, CRYA1, CRYBB2,CRYB2, PITX3, disorders BFSP2, CP49, CP47, CRYAA, CRYA1, PAX6, AN2,MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2,CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2,CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYA1,GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1); Cornealclouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2,TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2,PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma (MYOC,TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A,OPA1, NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1, RP12,CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D,GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4, ADMD,STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).

TABLE 5 SIGNAL TRANSDUCTION PATHWAYS Pathway Genes PI3K/AKT SignalingPRKCE; ITGAM; ITGA5; IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; PRKCZ; GRK6;MAPK1; TSC1; PLK1; AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2;PIK3CB; PPP2R1A; MAPK8; BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1;RELA; PRKCD; NOS3; PRKAA1; MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK;TP53; RAF1; IKBKG; RELB; DYRK1A; CDKN1A; ITGB1; MAP2K2; JAK1; AKT1;JAK2; PIK3R1; CHUK; PDPK1; PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3;CCND1; GSK3A; FRAP1; SFN; ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1;SGK; HSP90AA1; RPS6KB1 ERK/MAPK Signaling PRKCE; ITGAM; ITGA5; HSPB1;IRAK1; PRKAA2; EIF2AK2; RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1;RAC2; PLK1; AKT2; PIK3CA; CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4;PIK3CB; PPP2R1A; PIK3C3; MAPK8; MAPK3; ITGA1; ETS1; KRAS; MYCN;EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PPP2CA; PIM1; PIK3C2A;ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4;PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3; ITGB3; ESR1; ITGA2; MYC; TTK;CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF; STAT1; SGK Glucocorticoid RAC1;TAF4B; EP300; SMAD2; TRAF6; PCAF; ELK1; MAPK1; SMAD3; Receptor SignalingAKT2; IKBKB; NCOR2; UBE2I; PIK3CA; CREB1; FOS; HSPA5; NFKB2; BCL2;MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8; BCL2L1; MAPK3; TSC22D3; MAPK10;NRIP1; KRAS; MAPK13; RELA; STAT5A; MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A;CDKN1C; TRAF2; SERPINE1; NCOA3; MAPK14; TNF; RAF1; IKBKG; MAP3K7;CREBBP; CDKN1A; MAP2K2; JAK1; IL8; NCOA2; AKT1; JAK2; PIK3R1; CHUK;STAT3; MAP2K1; NFKB1; TGFBR1; ESR1; SMAD4; CEBPB; JUN; AR; AKT3; CCL2;MMP1; STAT1; IL6; HSP90AA1 Axonal Guidance PRKCE; ITGAM; ROCK1; ITGA5;CXCR4; ADAM12; IGF1; RAC1; RAP1A; Signaling E1F4E; PRKCZ; NRP1; NTRK2;ARHGEF7; SMO; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; AKT2; PIK3CA;ERBB2; PRKCI; PTK2; CFL1; GNAQ; PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1;GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PIK3C2A; ITGB7; GLI2;PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; ADAM17; AKT1; PIK3R1; GLI1;WNT5A; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; CRKL;RND1; GSK3B; AKT3; PRKCA Ephrin Receptor PRKCE; ITGAM; ROCK1; ITGA5;CXCR4; IRAK1; PRKAA2; EIF2AK2; Signaling RAC1; RAP1A; GRK6; ROCK2;MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1; AKT2; DOK1; CDK8; CREB1; PTK2;CFL1; GNAQ; MAP3K14; CXCL12; MAPK8; GNB2L1; ABL1; MAPK3; ITGA1; KRAS;RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PIM1; ITGB7; PXN; RAF1; FYN;DYRK1A; ITGB1; MAP2K2; PAK4, AKT1; JAK2; STAT3; ADAM10; MAP2K1; PAK3;ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK; CSNK1A1; CRKL; BRAF; PTPN13;ATF4; AKT3; SGK Actin Cytoskeleton ACTN4; PRKCE; ITGAM; ROCK1; ITGA5;IRAK1; PRKAA2; EIF2AK2; Signaling RAC1; INS; ARHGEF7; GRK6; ROCK2;MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1; PIK3CB; MYH9; DIAPH1;PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS; RHOA; PRKCD; PRKAA1;MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN; VIL2; RAF1; GSN; DYRK1A;ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1; PAK3; ITGB3; CDC42; APC;ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGK Huntington's Disease PRKCE;IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2; MAPK1; CAPNS1; Signaling AKT2;EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5; CREB1; PRKC1; HSPA5; REST; GNAQ;PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1; GNB2L1; BCL2L1; CAPN1; MAPK3;CASP8; HDAC2; HDAC7A; PRKCD; HDAC11; MAPK9; HDAC9; PIK3C2A; HDAC3; TP53;CASP9; CREBBP; AKT1; PIK3R1; PDPK1; CASP1; APAF1; FRAP1; CASP2; JUN;BAX; ATF4; AKT3; PRKCA; CLTC; SGK; HDAC6; CASP3 Apoptosis SignalingPRKCE; ROCK1; BID; IRAK1; PRKAA2; EIF2AK2; BAK1; BIRC4; GRK6; MAPK1;CAPNS1; PLK1; AKT2; IKBKB; CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14;MAPK8; BCL2L1; CAPN1; MAPK3; CASP8; KRAS; RELA; PRKCD; PRKAA1; MAPK9;CDK2; PIM1; TP53; TNF; RAF1; IKBKG; RELB; CASP9; DYRK1A; MAP2K2; CHUK;APAF1; MAP2K1; NFKB1; PAK3; LMNA; CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX;PRKCA; SGK; CASP3; BIRC3; PARP1 B Cell Receptor RAC1; PTEN; LYN; ELK1;MAPK1; RAC2; PTPN11; AKT2; IKBKB; Signaling PIK3CA; CREB1; SYK; NFKB2;CAMK2A; MAP3K14; PIK3CB; PIK3C3; MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS;MAPK13; RELA; PTPN6; MAPK9; EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG;RELB; MAP3K7; MAP2K2; AKT1; PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A;FRAP1; BCL6; BCL10; JUN; GSK3B; ATF4; AKT3; VAV3; RPS6KB1 LeukocyteACTN4; CD44; PRKCE; ITGAM; ROCK1; CXCR4; CYBA; RAC1; RAP1A;Extravasation Signaling PRKCZ; ROCK2; RAC2; PTPN11; MMP14; PIK3CA;PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8; PRKD1; ABL1; MAPK10; CYBB;MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A; BTK; MAPK14; NOX1; PXN; VIL2;VASP; ITGB1; MAP2K2; CTNND1; PIK3R1; CTNNB1; CLDN1; CDC42; F11R; ITK;CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9 Integrin Signaling ACTN4; ITGAM;ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1; ARHGEF7; MAPK1; RAC2; CAPNS1;AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3; MAPK8; CAV1; CAPN1; ABL1;MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7; PPP1CC; ILK; PXN; VASP;RAFI; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1; TNK2; MAP2K1; PAK3; ITGB3;CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3 Acute Phase Response IRAK1;SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11; AKT2; IKBKB; Signaling PIK3CA;FOS; NFKB2; MAP3K14; PIK3CB; MAPK8; RIPK1; MAPK3; IL6ST; KRAS; MAPK13;IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1; TRAF2; SERPINE1; MAPK14; TNF;RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1; CHUK;STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3; IL1R1; IL6 PTEN SignalingITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11; MAPK1; RAC2; AKT2; EGFR;IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2; PIK3CB; BCL2L1; MAPK3;ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1; IKBKG; CASP9; CDKN1A;ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1; MAP2K1; NFKB1; ITGB3;CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1; CASP3; RPS6KB1 p53Signaling PTEN; EP300; BBC3; PCAF; FASN; BRCA1; GADD45A; BIRC5; AKT2;PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3; MAPK8; THBS1; ATR;BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1; HDAC9; CDK2; PIK3C2A;MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1; RRM2B; APAF1; CTNNB1;SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2; GSK3B; BAχ; AKT3 ArylHydrocarbon HSPB1; EP300; FASN; TGM2; RXRA; MAPK1; NQO1; NCOR2; SP1;ARNT; Receptor Signaling CDKN1B; FOS; CHEK1; SMARCA4; NFKB2; MAPK8;ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73; GSTP1; RB1; SRC;CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2; APAF1; NFKB1; CCND1;ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; IL6; CYP1B1; HSP90AA1 XenobioticMetabolism PRKCE; EP300; PRKCZ; RXRA; MAPK1; NQO1; NCOR2; PIK3CA; ARNT;Signaling PRKCI; NFKB2; CAMK2A; PIK3CB; PPP2R1A; PIK3C3; MAPK8; PRKD1;ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13; PRKCD; GSTP1; MAPK9; NOS2A; ABCB1;AHR; PPP2CA; FTL; NFE2L2; PIK3C2A; PPARGC1A; MAPK14; TNF; RAF1; CREBBP;MAP2K2; PIK3R1; PPP2R5C; MAP2K1; NFKB1; KEAP1; PRKCA; EIF2AK3; IL6;CYP1B1; HSP90AA1 SAPK/JNK Signaling PRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1;ELK1; GRK6; MAPK1; GADD45A; RAC2; PLK1; AKT2; PIK3CA; FADD; CDK8;PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1; IRS1; MAPK3; MAPK10; DAXX; KRAS;PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; TRAF2; TP53; LCK; MAP3K7;DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3; CDC42; JUN; TTK; CSNK1A1; CRKL;BRAF; SGK PPAr/RXR Signaling PRKAA2; EP300; INS; SMAD2; TRAF6; PPARA;FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB; NCOR2; ABCA1; GNAQ; NFKB2;MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS; RELA; PRKAA1; PPARGC1A;NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; JAK2;CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1;ADIPOQ NF-KB Signaling IRAKI; EIF2AK2; EP300; INS; MYD88; PRKCZ: TRAF6;TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2; MAP3K14; PIK3CB; PIK3C3;MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A; TRAF2; TLR4: PDGFRB; TNF;INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1; PIK3R1; CHUK; PDGFRA;NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1 Neuregulin SignalingERBB4; PRKCE; ITGAM; ITGA5: PTEN; PRKCZ; ELK1; MAPK1; PTPN11; AKT2;EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3; ITGA1; KRAS; PRKCD;STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17; AKT1; PIK3R1; PDPK1;MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC; NRG1; CRKL; AKT3; PRKCA;HSP90AA1; RPS6KB1 Wnt & Beta catenin CD44; EP300; LRP6; DVL3; CSNK1E;GJA1; SMO; AKT2; PIN1; CDH1; Signaling BTRC; GNAQ; MARK2; PPP2R1A;WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2: ILK; LEF1; SOX9; TP53; MAP3K7;CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1; TGFBR1; CCND1;GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2 InsulinReceptor PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; PTPN11; AKT2; CBL;Signaling PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3; TSC2; KRAS;EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2; JAK1; AKT1;JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B; AKT3; FOXO1;SGK; RPS6KB1 IL-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1; MAPK1; PTPN11;IKBKB; FOS; NFKB2: MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST; KRAS; MAPK13;IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1; IKBKG; RELB;MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1; CEBPB; JUN;IL1R1; SRF; IL6 Hepatic Cholestasis PRKCE; IRAK1; INS; MYD88; PRKCZ;TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8; PRKD1; MAPK10;RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7;IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN; IL1R1; PRKCA;IL6 IGF-1 Signaling IGF1; PRKCZ; ELK1; MAPK1; PTPN11; NEDD4; AKT2;PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R; IRS1; MAPK3;IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2; AKT1; PIK3R1;PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF; CTGF; RPS6KB1NRF2-mediated PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1; NQO1; PIK3CA;PRKCI; Oxidative Stress FOS; PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3; KRAS;PRKCD; GSTP1; Response MAPK9; FTL; NFE2L2; PIK3C2A; MAPK14; RAF1;MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN; KEAP1; GSK3B;ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic EDN1; IGF1; KDR; FLT1; SMAD2;FGFR1; MET; PGF; SMAD3; EGFR; Fibrosis/Hepatic FAS; CSF1; NFKB2; BCL2;MYH9; IGF1R; IL6R; RELA; TLR4; PDGFRB; Stellate Cell Activation TNF;RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1; CCL2; HGF;MMP1; STAT1; IL6; CTGF; MMP9 PPAR Signaling EP300; INS; TRAF6; PPARA;RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3; NRIP1;KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF; INSR; RAF1;IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1; NFKB1; JUN;IL1R1; HSP90AA1 Fc Epsilon RI Signaling PRKCE; RAC1; PRKCZ; LYN; MAPK1;RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; MAPK8; PRKD1;MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK; MAPK14; TNF;RAF1; FYN; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; VAV3; PRKCAG-Protein Coupled PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB; PIK3CA;CREB1; Receptor Signaling GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3; MAPK3;KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1; PIK3R1;CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCA InositolPhosphate PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; MAPK1; PLK1; AKT2;Metabolism PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD; PRKAA1;MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1; MAP2K1;PAK3; ATM; TTK; CSNK1A1; BRAF; SGK PDGF Signaling EIF2AK2; ELK1; ABL2;MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3; KRAS; SRC;PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA; STAT3; SPHK1;MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGF Signaling ACTN4;ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA; ARNT; PTK2; BCL2;PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN; RAF1;MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA; AKT3; FOXO1; PRKCANatural Killer Cell PRKCE; RAC1; PRKCZ; MAPK1; RAC2; PTPN11; KIR2DL3;AKT2; Signaling PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; PRKD1; MAPK3; KRAS;PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4; AKT1; PIK3R1;MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: G1/S HDAC4; SMAD3; SUV39H1;HDAC5; CDKN1B; BTRC; ATR; ABL1; E2F1; Checkpoint Regulation HDAC2;HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1;E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1; HDAC6 T CellReceptor RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS; NFKB2; PIK3CB;Signaling PIK3C3; MAPK8; MAPK3; KRAS; RELA, PIK3C2A; BTK; LCK; RAF1;IKBKG; RELB, FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK; BCL10; JUN;VAV3 Death Receptor CRADD; HSPB1; BID; BIRC4; TBK1; IKBKB; FADD; FAS;NFKB2; BCL2; Signaling MAP3K14; MAPK8; RIPK1; CASP8; DAXX; TNFRSF10B;RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1; CASP2; BIRC2;CASP3; BIRC3 FGF Signaling RAC1; FGFR1; MET; MAPKAPK2; MAPK1; PTPN11;AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3; MAPK13; PTPN6;PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1; FGFR4; CRKL; ATF4;AKT3; PRKCA; HGF GM-CSF Signaling LYN; ELK1; MAPK1; PTPN11; AKT2;PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1; MAPK3; ETS1;KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2; PIK3R1; STAT3;MAP2K1; CCND1; AKT3; STAT1 Amyotrophic Lateral BID; IGF1; RAC1; BIRC4;PGF; CAPNS1; CAPN2; PIK3CA; BCL2; PIK3CB; Sclerosis Signaling PIK3C3;BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1; APAF1; VEGFA;BIRC2; BAχ; AKT3; CASP3; BIRC3 JAK/Stat Signaling PTPN1; MAPK1; PTPN11;AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS; SOCS1; STAT5A; PTPN6;PIK3C2A; RAFI; CDKN1A; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; STAT3; MAP2K1;FRAP1; AKT3; STAT1 Nicotinate and PRKCE; IRAK1; PRKAA2; EIF2AK2; GRK6;MAPK1; PLK1; AKT2; CDK8; Nicotinamide MAPK8; MAPK3; PRKCD; PRKAA1;PBEF1; MAPK9; CDK2; PIM1; Metabolism DYRK1A; MAP2K2; MAP2K1; PAK3; NT5E;TTK; CSNK1A1; BRAF; SGK Chemokine Signaling CXCR4; ROCK2; MAPK1; PTK2;FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8; MAPK3; KRAS; MAPK13; RHOA; CCR3;SRC; PPP1CC; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1; JUN; CCL2; PRKCA IL-2Signaling ELK1; MAPK1; PTPN11; AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB;PIK3C3; MAPK8; MAPK3; KRAS; SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2;JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3 Synaptic Long Term PRKCE; IGF1;PRKCZ; PRDX6; LYN; MAPK1; GNAS; PRKCI; GNAQ; Depression PPP2R1A; IGF1R;PRKD1; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1;MAP2K2; PPP2R5C; MAP2K1; PRKCA Estrogen Receptor TAF4B; EP300; CARM1;PCAF; MAPK1; NCOR2; SMARCA4; MAPK3; Signaling NRIP1; KRAS; SRC; NR3C1;HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1;PRKDC; ESR1; ESR2 Protein Ubiquitination TRAF6; SMURF1; BIRC4; BRCA1;UCHL1; NEDD4; CBL; UBE2I; BTRC; Pathway HSPA5; USP7; USP10; FBXW7;USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1; VHL; HSP90AA1; BIRC3IL-10 Signaling TRAF6; CCR1; ELK1; IKBKB; SP1; FOS; NFKB2; MAP3K14;MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB; MAP3K7; JAK1; CHUK;STAT3; NFKB1; JUN; IL1R1; IL6 VDR/RXR Activation PRKCE; EP300; PRKCZ;RXRA; GADD45A; HES1; NCOR2; SP1; PRKCI; CDKN1B; PRKD1; PRKCD; RUNX2;KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1; LRP5; CEBPB; FOXO1; PRKCATGF-beta Signaling EP300; SMAD2; SMURF1; MAPK1; SMAD3; SMAD1; FOS;MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1; RAF1; MAP3K7; CREBBP;MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5 Toll-like Receptor IRAKI;EIF2AK2; MYD88; TRAF6; PPARA; ELK1; IKBKB; FOS; NFKB2; SignalingMAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK;NFKB1; TLR2; JUN p38 MAPK Signaling HSPB1; IRAK1; TRAF6; MAPKAPK2; ELK1;FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK14; TNF;MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1 Neurotrophin/TRK NTRK2;MAPK1; PTPN11; PIK3CA; CREB1; FOS; PIK3CB; PIK3C3; Signaling MAPK8;MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42;JUN; ATF4 FXR/RXR Activation INS; PPARA; FASN; RXRA; AKT2; SDC1; MAPK8;APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; TNF; CREBBP; AKT1; SREBF1;FGFR4; AKT3; FOXO1 Synaptic Long Term PRKCE; RAP1A; EP300; PRKCZ; MAPK1;CREB1; PRKCI; GNAQ; Potentiation CAMK2A; PRKD1; MAPK3; KRAS; PRKCD;PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4; PRKCA Calcium SignalingRAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1; CAMK2A; MYH9; MAPK3; HDAC2;HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4; HDAC6 EGFSignaling ELK1; MAPK1; EGFR; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3;PIK3C2A; RAFI; JAK1; PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1Hypoxia Signaling in EDN1; PTEN; EP300; NQO1; UBE2I; CREB1; ARNT; HIF1A;SLC2A4; the Cardiovascular NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN;ATF4; VHL; HSP90AA1 System LPS/IL-1 Mediated IRAK1; MYD88; TRAF6; PPARA;RXRA; ABCA1, MAPK8; ALDH1A1; Inhibition of RXR GSTP1; MAPK9; ABCB1;TRAF2; TLR4; TNF; MAP3K7; NR1H2; SREBF1; Function JUN; IL1R1 LXR/RXRActivation FASN; RXRA; NCOR2; ABCA1; NFKB2; IRF3; RELA; NOS2A; TLR4;TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1; IL1R1; CCL2; IL6; MMP9 AmyloidProcessing PRKCE; CSNK1E; MAPK1; CAPNS1; AKT2; CAPN2; CAPN1; MAPK3;MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1; GSK3B; AKT3; APP IL-4Signaling AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1; KRAS; SOCS1; PTPN6; NR3C1;PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1; AKT3; RPS6KB1 Cell Cycle: G2/MDNA EP300; PCAF; BRCA1; GADD45A; PLK1; BTRC; CHEK1; ATR; CHEK2; DamageCheckpoint YWHAZ; TP53; CDKN1A; PRKDC; ATM; SFN; CDKN2A RegulationNitric Oxide Signaling KDR; FLT1; PGF; AKT2; PIK3CA; PIK3CB; PIK3C3;CAV1; PRKCD; NOS3; in the Cardiovascular PIK3C2A; AKT1; PIK3R1; VEGFA;AKT3; HSP90AA1 System Purine Metabolism NME2; SMARCA4; MYH9; RRM2; ADAR;EIF2AK4; PKM2; ENTPD1; RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; NME1cAMP-mediated RAP1A; MAPK1; GNAS; CREB1; CAMK2A; MAPK3; SRC; RAF1;Signaling MAP2K2; STAT3; MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8;CASP8; MAPK10; MAPK9; CASP9; PARK7; PSEN1; Dysfunction PARK2; APP; CASP3Notch Signaling HES1; JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3;NOTCH1; DLL4 Endoplasmic Reticulum HSPA5; MAPK8; XBP1; TRAF2; ATF6;CASP9; ATF4; EIF2AK3; CASP3 Stress Pathway Pyrimidine Metabolism NME2;AICDA; RRM2; EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson'sSignaling UCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; CASP3Cardiac & Beta GNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; PPP2R5CAdrenergic Signaling Glycolysis/ HK2; GCK; GPI; ALDH1A1; PKM2; LDHA; HK1Gluconeogenesis Interferon Signaling IRF1; SOCS1; JAK1; JAK2; IFITM1;STAT1; IFIT3 Sonic Hedgehog ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B;DYRKIB Signaling Glycerophospholipid PLD1; GRN; GPAM; YWHAZ; SPHK1;SPHK2 Metabolism Phospholipid PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2Degradation Tryptophan Metabolism SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1;SIAH1 Lysine Degradation SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C NucleotideExcision ERCC5; ERCC4; XPA; XPC; ERCC1 Repair Pathway Starch and SucroseUCHL1; HK2; GCK; GPI; HK1 Metabolism Aminosugars NQO1; HK2; GCK; HK1Metabolism Arachidonic Acid PRDX6; GRN; YWHAZ; CYP1B1 MetabolismCircadian Rhythm CSNK1E; CREB1; ATF4; NR1D1 Signaling Coagulation SystemBDKRB1; F2R; SERPINE1; F3 Dopamine Receptor PPP2R1A; PPP2CA; PPP1CC;PPP2R5C Signaling Glutathione IDH2; GSTP1; ANPEP; IDH1 MetabolismGlycerolipid ALDH1A1; GPAM; SPHK1; SPHK2 Metabolism Linoleic Acid PRDX6;GRN; YWHAZ; CYP1B1 Metabolism Methionine Metabolism DNMT1; DNMT3B; AHCY;DNMT3A Pyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA Arginine andProline ALDH1A1; NOS3; NOS2A Metabolism Eicosanoid Signaling PRDX6; GRN;YWHAZ Fructose and Mannose HK2; GCK; HK1 Metabolism Galactose MetabolismHK2; GCK; HK1 Stilbene, Coumarine PRDX6; PRDX1; TYR and LigninBiosynthesis Antigen Presentation CALR; B2M Pathway Biosynthesis ofSteroids NQO1; DHCR7 Butanoate Metabolism ALDH1A1; NLGN1 Citrate CycleIDH2; IDH1 Fatty Acid Metabolism ALDH1A1; CYP1B1 GlycerophospholipidPRDX6; CHKA Metabolism Histidine Metabolism PRMT5; ALDH1A1 InositolMetabolism ERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics byCytochrome p450 Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6;PRDX1 Metabolism Propanoate Metabolism ALDH1A1; LDHA Selenoamino AcidPRMT5; AHCY Metabolism Sphingolipid SPHK1; SPHK2 MetabolismAminophosphonate PRMT5 Metabolism Androgen and Estrogen PRMT5 MetabolismAscorbate and Aldarate ALDH1A1 Metabolism Bile Acid Biosynthesis ALDH1A1Cysteine Metabolism LDHA Fatty Acid Biosynthesis FASN Glutamate ReceptorGNB2L1 Signaling NRF2-mediated PRDX1 Oxidative Stress Response PentosePhosphate GPI Pathway Pentose and UCHL1 Glucuronate InterconversionsRetinol Metabolism ALDH1A1 Riboflavin Metabolism TYR Tyrosine MetabolismPRMT5, TYR Ubiquinone PRMT5 Biosynthesis Valine, Leucine and ALDH1A1Isoleucine Degradation Glycine, Serine and CHKA Threonine MetabolismLysine Degradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain TRPM7; TRPC5;TRPC6; TRPC1; Cnr1; cm2; Grk2; Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b;TRPM5; Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial Function AIF;CytC; SMAC (Diablo); Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd);Noggin (Nog); WNT (Wnt2; Wnt2b; Wnt3a; Wnt4; Neurology Wnt5a; Wnt6;Wnt7b; Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta- catenin;Dkk-1; Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1;unc-86 (Pou4fl or Bm3a); Numb; RelnViral Vectors

There are provided, in some embodiments, viral vectors. The viral vectorcan be an RNA viral vector. The polynucleotide can be derived from apositive sense RNA virus, a negative sense RNA virus, an ambisense RNAvirus, or any combination thereof. The polynucleotide can be derivedfrom a single-stranded RNA virus. The polynucleotide can be derived froma negative-strand RNA virus. The polynucleotide can be derived from oneor more negative-strand RNA viruses of the order Mononegavirales. Thenucleoprotein (N), phosphoprotein (P), matrix protein (M), and/orRNA-dependent RNA polymerase (L) can be derived from one or morenegative-strand RNA viruses of the order Mononegavirales (e.g., abornaviridae virus, a filoviridae virus, a nyamiviridae virus, aparamyxodiridae virus, a rhabdoviridae virus, or any combinationthereof). The Mononegavirales virus can comprise rabies virus, sendaivirus, vesicular stomatitis virus, or any combination thereof. AMononegavirales-based viral vector can comprise one or more attenuatingmutations. In some embodiments, the one or more negative-strand RNAviruses of the order Mononegavirales can comprise an attenuated rabiesvirus strain (e.g., CVS-N2c, CVS-B2c, DRV-4, RRV-27, SRV-16, ERA,CVS-11, SAD B19, SPBN, SN-10, SN10-333, PM, LEP, SAD, or any combinationthereof).

Viral vectors and methods of using are provided in PCT ApplicationPublication No. WO2020/210655A1 and U.S. Patent Publication No.2020/0165576, the content of each of which is incorporated herein byreference in its entirety. The viral vector can be modified so that theviral vector is targeted to a particular target environment of interestsuch as central nervous system, and to enhance tropism to the targetenvironment of interest (e.g, CNS tropism). In some embodiments, theviral vector is AAV-CAP.B22. Broad gene expression throughout the mouseand marmoset brain after intravenous delivery of engineered AAV capsidshas been described in Flytzanis et al. (“Broad gene expressionthroughout the mouse and marmoset brain after intravenous delivery ofengineered AAV capsids” biorxiv, 2020), the content of which isincorporated herein by reference in its entirety.

Exemplary viral vectors that can be used in the methods, compositions,systems and kits described herein include those provided in US20200071723A1, the content of which is incorporated herein by referencein its entirety. In some embodiments, the vector can comprise anadenovirus vector, an adeno-associated virus vector (AAV), anEpstein-Barr virus vector, a Herpes virus vector, an attenuated HIVvector, a retroviral vector, a vaccinia virus vector, or any combinationthereof. In some embodiments, the vector can comprise an RNA viralvector. In some embodiments, the vector can be derived from one or morenegative-strand RNA viruses of the order Mononegavirales. In someembodiments, the vector can be a rabies viral vector. Many such vectorsuseful for transferring exogenous genes into target mammalian cells areavailable. The vectors may be episomal, e.g. plasmids, virus-derivedvectors such cytomegalovirus, adenovirus, etc., or may be integratedinto the target cell genome, through homologous recombination or randomintegration, e.g. retrovirus-derived vectors such as MMLV, HIV-1, ALV,etc. In some embodiments, combinations of retroviruses and anappropriate packaging cell line may also find use, where the capsidproteins will be functional for infecting the target cells. Retroviralvectors can be “defective”, i.e. unable to produce viral proteinsrequired for productive infection. Replication of the vector can requiregrowth in the packaging cell line. The term “vector”, as used herein,refers to a nucleic acid construct designed for delivery to a host cellor for transfer between different host cells. As used herein, a vectorcan be viral or non-viral. The term “vector” encompasses any geneticelement that is capable of replication when associated with the propercontrol elements and that can transfer gene sequences to cells. A vectorcan include, but is not limited to, a cloning vector, an expressionvector, a plasmid, phage, transposon, cosmid, artificial chromosome,virus, virion, etc.

In some embodiment, the vectors can include a regulatory sequence thatallows, for example, the translation of multiple proteins from a singlemRNA. Non-limiting examples of such regulatory sequences includeinternal ribosome entry site (IRES) and 2A self-processing sequence. Insome embodiments, the 2A sequence is a 2A peptide site fromfoot-and-mouth disease virus (F2A sequence). In some embodiments, theF2A sequence has a standard furin cleavage site. In some embodiments,the vector can also comprise regulatory control elements known to one ofskill in the art to influence the expression of the RNA and/or proteinproducts encoded by the polynucleotide within desired cells of thesubject. In some embodiments, functionally, expression of thepolynucleotide is at least in part controllable by the operably linkedregulatory elements such that the element(s) modulates transcription ofthe polynucleotide, transport, processing and stability of the RNAencoded by the polynucleotide and, as appropriate, translation of thetranscript. A specific example of an expression control element is apromoter, which is usually located 5′ of the transcribed sequence.Another example of an expression control element is an enhancer, whichcan be located 5′ or 3′ of the transcribed sequence, or within thetranscribed sequence. Another example of a regulatory element is arecognition sequence for a microRNA. Another example of a regulatoryelement is an ration and the splice donor and splice acceptor sequencesthat regulate the splicing of said intron. Another example of aregulatory element is a transcription termination signal and/or apolyadenylation sequence.

Expression control elements and promoters include those active in aparticular tissue or cell type, referred to herein as a “tissue-specificexpression control elements/promoters.” Tissue-specific expressioncontrol elements are typically active in specific cell or tissue (forexample in the liver, brain, central nervous system, spinal cord, eye,retina or lung). Expression control elements are typically active inthese cells, tissues or organs because they are recognized bytranscriptional activator proteins, or other regulators oftranscription, that are unique to a specific cell, tissue or organ type.

Expression control elements also include ubiquitous or promiscuouspromoters/enhancers which are capable of driving expression of apolynucleotide in many different cell types. Such elements include, butare not limited, to the cytomegalovirus (CMV) immediate earlypromoter/enhancer sequences, the Rous sarcoma virus (RSV)promoter/enhancer sequences and the other viral promoters/enhancersactive in a variety of mammalian cell types; promoter/enhancer sequencesfrom ubiquitously or promiscuously expressed mammalian genes including,but not limited to, beta actin, ubiquitin or EF1 alpha; or syntheticelements that are not present in nature.

Expression control elements also can confer expression in a manner thatis regulatable, that is, a signal or stimuli increases or decreasesexpression of the operably linked polynucleotide. A regulatable elementthat increases expression of the operably linked polynucleotide mresponse to a signal or stimuli is also referred to as an “inducibleelement” (that is, it is induced by a signal). Particular examplesinclude, but are not limited to, a hormone (for example, steroid)inducible promoter. A regulatable element that decreases expression ofthe operably linked polynucleotide in response to a signal or stimuli isreferred to as a “repressible element” (that is, the signal decreasesexpression such that when the signal, is removed or absent, expressionis increased). Typically, the amount of increase or decrease conferredby such elements is proportional to the amount of signal or stimulipresent: the greater the amount of signal or stimuli, the greater theincrease or decrease in expression.

Methods of Detecting and Monitoring

In some embodiments, the methods and compositions provided herein areuseful in detecting a disease or disorder and/or monitoring theprogression of a disease or disorder. As used herein, the term“diagnostic” refers identifying the presence or absence of or nature ofa disease or disorder. Such detection methods can be used, for example,for early diagnosis of the condition, to determine whether a subject ispredisposed to a disease or disorder, to monitor the progress of thedisease or disorder or the progress of treatment protocols, to assessthe severity of the disease or disorder, to forecast the an outcome of adisease or disorder and/or prospects of recovery, or to aid in thedetermination of a suitable treatment for a subject. The detection canoccur in vitro or in vivo. The payload protein can comprise a diagnosticagent. The payload protein can comprise a diagnostic contrast agent. Thediagnostic agent can comprise green fluorescent protein (GFP), enhancedgreen fluorescent protein (EGFP), yellow fluorescent protein (YFP),enhanced yellow fluorescent protein (EYFP), blue fluorescent protein(BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple,mCherry, mruby3, rsCherry, rsCherryRev, derivatives thereof, or anycombination thereof.

In some embodiments, the payload protein encodes a diagnostic agent. Insome embodiments, the diagnostic agent aids in the identification of aunique cell type and/or a unique cell state. The diagnostic agent can bea molecule capable of detection, including, but not limited to,fluorescers, chemiluminescers, chromophores, bioluminescent proteins,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,isotopic labels, semiconductor nanoparticles, dyes, metal ions, metalsols, ligands (e.g., biotin, streptavidin or haptens) and the like. Theterm “fluorescer” refers to a substance or a portion thereof which iscapable of exhibiting fluorescence in the detectable range. For example,the diagnostic agent may comprise, in some embodiments, a fluorescentprotein, such as, but not limited to, green fluorescent protein (GFP),enhanced green fluorescent protein (EGFP), yellow fluorescent protein(YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescentprotein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron,mApple, mCherry, rsCherry, rsCherryRev, or any combination thereof. Insome embodiments, the expression, stability, and/or activity (e.g.,fluorescence) of the diagnostic agent is configured to be responsive toa disease state or a disorder state.

In some embodiments, the diagnostic agent aids in the identification ofa unique cell type and/or a unique cell state. The unique cell typeand/or a unique cell state can comprise lesions (e.g. tumors, infectedcells). Detection and/or imaging of the diagnostic agent can enable aclinician to intraoperatively, laparoscopically, intravascularly orendoscopically detect said lesions. In some such embodiments,discrimination between lesions (e.g. tumors) and non-lesions (e.g.non-tumor tissue) is enhanced by the detection and/or imaging of thediagnostic agent. In some embodiments, detection and/or imaging of thediagnostic agent can enable a clinician to accurately locate lesions ina patient and thereby aid resection, irradiation, biopsy and/or lesionremoval. In some embodiments, detection and/or imaging of the diagnosticagent aids the detection of non-malignant pathological lesions, such as,an infarct, including myocardial, atherosclerotic plaque, clot,including thrombosis, pulmonary embolism, infectious or inflammatorylesion, non-tumorous or noninfectious inflammation, or hyperplasia. Thedetection and/or imaging of the diagnostic agent may also be used todetect various stages of progression or severity of disease (e.g.,benign, premalignant, and malignant breast lesions, tumor growth, ormetastasis). The detection and/or imaging of the diagnostic agent mayalso be used to detect the response of the disease to prophylactic ortherapeutic treatments or other interventions. The detection and/orimaging of the diagnostic agent can furthermore be used to help themedical practitioner in determining prognosis (e.g., worsening,status-quo, partial recovery, or complete recovery) of the patient, andthe appropriate course of action.

Detection and/or imaging of the diagnostic agent can be performed, forexample, using an ultrasound scanner, a magnetic resonance imaginginstrument (MRI scanner), an X-ray source with film or a detector (e.g.,conventional or digital radiography system), an X-ray computedtomography (CT) or computed axial tomography (CAT) scanner, a gammacamera, or a positron emission tomography (PET) scanner. Various medicalimaging systems have been developed for open surgery as well as forlaparoscopic, thoracoscopic, and robot-assisted surgery and can be usedin the practice of the invention. Conventional laparoscopes andendoscopes can be equipped with a photodetector (e.g., camera or CCDdetector) to provide guidance during medical procedures. Fiber-opticimaging systems can also be used, which include portable handheldmicroscopes, flexible endoscopes, and microendoscopes. For example, anillumination source can be added to such devices to allow fluorescenceimaging. A miniaturized ultrasound transducer can be added to the tip ofa laparoscope or catheter for intravascular ultrasound (IVUS) imaging.Miniaturized imaging systems can be used that allow imaging inside smallcavities and constricted spaces. In addition, miniaturized imagingdevices (e.g., microendoscopes) may be implanted within a subject forlong-term imaging studies. In addition, a camera may be used to takeboth photographic images of a subject and to detect signals from thediagnostic agent, so that photographic images of the subject and imagesof the signals from the diagnostic agent can be superimposed to allowregions containing the diagnostic agent to be mapped to the subject'sanatomy.

Pharmaceutical Compositions and Methods of Administration

Also disclosed herein are pharmaceutical compositions comprising one ormore of the nucleic acids, vectors, and/or compositions provided hereinand one or more pharmaceutically acceptable carriers. The compositionscan also comprise additional ingredients such as diluents, stabilizers,excipients, and adjuvants. As used herein, “pharmaceutically acceptable”carriers, excipients, diluents, adjuvants, or stabilizers are the onesnontoxic to the cell or subject being exposed thereto (preferably inert)at the dosages and concentrations employed or that have an acceptablelevel of toxicity as determined by the skilled practitioners.

The carriers, diluents and adjuvants can include buffers such asphosphate, citrate, or other organic acids: antioxidants such asascorbic acid; low molecular weight polypeptides (e.g., less than about10 residues); proteins such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine, or lysine;monosaccharides, di saccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween™, Pluronics™ orpolyethylene glycol (PEG). In some embodiments, the physiologicallyacceptable carrier is an aqueous pH buffered solution.

Titers of vectors to be administered will vary depending, for example,on the particular viral vector, the mode of administration, thetreatment goal, the individual, and the cell type(s) being targeted, andcan be determined by methods standard in the art.

As will be readily apparent to one skilled in the art, the useful invivo dosage of the nucleic acids, vectors, and/or compositions to beadministered and the particular mode of administration will varydepending upon the age, weight, the severity of the affliction, andanimal species treated, the particular IFFL that is used, and thespecific use for which the IFFL is employed. The determination ofeffective dosage levels, that is the dosage levels necessary to achievethe desired result, can be accomplished by one skilled in the art usingroutine pharmacological methods. Typically, human clinical applicationsof products are commenced at lower dosage levels, with dosage levelbeing increased until the desired effect is achieved. Alternatively,acceptable in vitro studies can be used to establish useful doses androutes of administration of the compositions identified by the presentmethods using established pharmacological methods.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made.Dosages of nucleic acids, vectors, and/or compositions provided candepend primarily on factors such as the condition being treated, theage, weight and health of the patient, and may thus vary among patients.For example, a therapeutically effective human dosage of the viralvector is generally in the range of from about 0.1 ml to about 100 ml ofsolution containing concentrations of from about 1×10⁹ to 1×10¹⁶ genomesvirus viral. A preferred human dosage can be about 1×10¹³ to 1×10¹⁶viral vector genomes. The dosage will be adjusted to balance thetherapeutic benefit against any side effects and such dosages may varydepending upon the therapeutic application for which the recombinantvector is employed. The levels of expression of the payload can bemonitored to determine the amount and/or frequency of dosage resultingfrom the viral vector in some embodiments.

Nucleic acids, vectors, and/or compositions disclosed herein can beadministered to a subject (e.g., a human) in need thereof. The route ofthe administration is not particularly limited. For example, atherapeutically effective amount of nucleic acids, vectors, and/orcompositions can be administered to the subject by via routes standardin the art. Route(s) of administration can be readily determined by oneskilled in the art taking into account the infection and/or diseasestate being treated and the target cells/tissue(s) that are to expressthe payload protein.

The administering can comprise systemic administration (e.g.,intravenous, intramuscular, intraperitoneal, or intraarticular).Administering can comprise intrathecal administration, intracranialinjection, aerosol delivery, nasal delivery, vaginal delivery, rectaldelivery, buccal delivery, ocular delivery, local delivery, topicaldelivery, intracisternal delivery, intraperitoneal delivery, oraldelivery, intramuscular injection, intravenous injection, subcutaneousinjection, intranodal injection, intratumoral injection, intraperitonealinjection, intradermal injection, or any combination thereof.

Administering can comprise an injection into a brain region (e.g.,direct administration to the brain parenchyma). The brain region cancomprise the Lateral parabrachial nucleus, brainstem, Medulla oblongata,Medullary pyramids, Olivary body, Inferior olivary nucleus, Rostralventrolateral medulla, Respiratory center, Dorsal respiratory group,Ventral respiratory group, Pre-Botzinger complex, Botzinger complex,Paramedian reticular nucleus, Cuneate nucleus, Gracile nucleus,Intercalated nucleus, Area postrema, Medullary cranial nerve nuclei,Inferior salivatory nucleus, Nucleus ambiguus, Dorsal nucleus of vagusnerve, Hypoglossal nucleus, Solitary nucleus, Pons, Pontine nuclei,Pontine cranial nerve nuclei, chief or pontine nucleus of the trigeminalnerve sensory nucleus (V), Motor nucleus for the trigeminal nerve (V),Abducens nucleus (VI), Facial nerve nucleus (VII), vestibulocochlearnuclei (vestibular nuclei and cochlear nuclei) (VIII), Superiorsalivatory nucleus, Pontine tegmentum, Respiratory centers, Pneumotaxiccenter, Apneustic center, Pontine micturition center (Barrington'snucleus), Locus coeruleus, Pedunculopontine nucleus, Laterodorsaltegmental nucleus, Tegmental pontine reticular nucleus, Superior olivarycomplex, Paramedian pontine reticular formation, Cerebellar peduncles,Superior cerebellar peduncle, Middle cerebellar peduncle, Inferiorcerebellar peduncle, Cerebellum, Cerebellar vermis, Cerebellarhemispheres, Anterior lobe, Posterior lobe, Flocculonodular lobe,Cerebellar nuclei, Fastigial nucleus, Interposed nucleus, Globosenucleus, Emboliform nucleus, Dentate nucleus, Tectum, Corporaquadrigemina, inferior colliculi, superior colliculi, Pretectum,Tegmentum, Periaqueductal gray, Parabrachial area, Medial parabrachialnucleus, Subparabrachial nucleus (Kolliker-Fuse nucleus), Rostralinterstitial nucleus of medial longitudinal fasciculus, Midbrainreticular formation, Dorsal raphe nucleus, Red nucleus, Ventraltegmental area, Substantia nigra, Pars compacta, Pars reticulata,Interpeduncular nucleus, Cerebral peduncle, Crus cerebri, Mesencephaliccranial nerve nuclei, Oculomotor nucleus (III), Trochlear nucleus (IV),Mesencephalic duct (cerebral aqueduct, aqueduct of Sylvius), Pinealbody, Habenular nucleim Stria medullares, Taenia thalami, Subcommissuralorgan, Thalamus, Anterior nuclear group, Anteroventral nucleus (akaventral anterior nucleus), Anterodorsal nucleus, Anteromedial nucleus,Medial nuclear group, Medial dorsal nucleus, Midline nuclear group,Paratenial nucleus, Reuniens nucleus, Rhomboidal nucleus, Intralaminarnuclear group, Centromedial nucleus, Parafascicular nucleus, Paracentralnucleus, Central lateral nucleus, Central medial nucleus, Lateralnuclear group, Lateral dorsal nucleus, Lateral posterior nucleus,Pulvinar, Ventral nuclear group, Ventral anterior nucleus, Ventrallateral nucleus, Ventral posterior nucleus, Ventral posterior lateralnucleus, Ventral posterior medial nucleus, Metathalamus, Medialgeniculate body, Lateral geniculate body, Thalamic reticular nucleus,Hypothalamus, limbic system, HPA axis, preoptic area, Medial preopticnucleus, Suprachiasmatic nucleus, Paraventricular nucleus, Supraopticnucleusm Anterior hypothalamic nucleus, Lateral preoptic nucleus, medianpreoptic nucleus, periventricular preoptic nucleus, Tuberal, Dorsomedialhypothalamic nucleus, Ventromedial nucleus, Arcuate nucleus, Lateralarea, Tuberal part of Lateral nucleus, Lateral tuberal nuclei,Mammillary nuclei, Posterior nucleus, Lateral area, Optic chiasm,Subfornical organ, Periventricular nucleus, Pituitary stalk, Tubercinereum, Tuberal nucleus, Tuberomammillary nucleus, Tuberal region,Mammillary bodies, Mammillary nucleus, Subthalamus, Subthalamic nucleus,Zona incerta, Pituitary gland, neurohypophysis, Pars intermedia,adenohypophysis, cerebral hemispheres, Corona radiata, Internal capsule,External capsule, Extreme capsule, Arcuate fasciculus, Uncinatefasciculus, Perforant Path, Hippocampus, Dentate gyms, Cornu ammonis,Cornu ammonis area 1, Cornu ammonis area 2, Cornu ammonis area 3, Cornuammonis area 4, Amygdala, Central nucleus, Medial nucleus (accessoryolfactory system), Cortical and basomedial nuclei, Lateral andbasolateral nuclei, extended amygdala, Stria terminalis, Bed nucleus ofthe stria terminalis, Claustrum, Basal ganglia, Striatum, Dorsalstriatum (aka neostriatum), Putamen, Caudate nucleus, Ventral striatum,Striatum, Nucleus accumbens, Olfactory tubercle, Globus pallidus,Subthalamic nucleus, Basal forebrain, Anterior perforated substance,Substantia innominata, Nucleus basalis, Diagonal band of Broca, Septalnuclei, Medial septal nuclei, Lamina terminalis, Vascular organ oflamina terminalis, Olfactory bulb, Piriform cortex, Anterior olfactorynucleus, Olfactory tract, Anterior commissure, Uncus, Cerebral cortex,Frontal lobe, Frontal cortex, Primary motor cortex, Supplementary motorcortex, Premotor cortex, Prefrontal cortex, frontopolar cortex,Orbitofrontal cortex, Dorsolateral prefrontal cortex, dorsomedialprefrontal cortex, ventrolateral prefrontal cortex, Superior frontalgyms, Middle frontal gyms, Inferior frontal gyms, Brodmann areas (4, 6,8, 9, 10, 11, 12, 24, 25, 32, 33, 44, 45, 46, and/or 47), Parietal lobe,Parietal cortex, Primary somatosensory cortex (S), Secondarysomatosensory cortex (S2), Posterior parietal cortex, postcentral gyms,precuneus, Brodmann areas (1, 2, 3 (Primary somesthetic area), 5, 7, 23,26, 29, 31, 39, and/or 40), Occipital lobe, Primary visual cortex (V1),V2, V3, V4, V5/MT, Lateral occipital gyms, Cuneus, Brodmann areas (17(V1, primary visual cortex), 18, and/or 19), temporal lobe, Primaryauditory cortex (A1), secondary auditory cortex (A2), Inferior temporalcortex, Posterior inferior temporal cortex, Superior temporal gyms,Middle temporal gyms, Inferior temporal gyms, Entorhinal Cortex,Perirhinal Cortex, Parahippocampal gyms, Fusiform gyms, Brodmann areas(9, 20, 21, 22, 27, 34, 35, 36, 37, 38, 41, and/or 42), Medial superiortemporal area (MST), insular cortex, cingulate cortex, Anteriorcingulate, Posterior cingulate, dorsal cingulate, Retrosplenial cortex,Indusium griseum, Subgenual area 25, Brodmann areas (23, 24; 26, 29, 30(retrosplenial areas), 31, and/or 32), cranial nerves (Olfactory (I),Optic (II), Oculomotor (III), Trochlear (IV), Trigeminal (V), Abducens(VI), Facial (VII), Vestibulocochlear (VIII), Glossopharyngeal (IX),Vagus (X), Accessory (XI), Hypoglossal (XII)), or any combinationthereof. The brain region can comprise neural pathways Superiorlongitudinal fasciculus, Arcuate fasciculus, Thalamocortical radiations,Cerebral peduncle, Corpus callosum, Posterior commissure, Pyramidal orcorticospinal tract, Medial longitudinal fasciculus, dopamine system,Mesocortical pathway, Mesolimbic pathway, Nigrostriatal pathway,Tuberoinfundibular pathway, serotonin system, Norepinephrine Pathways,Posterior column-medial lemniscus pathway, Spinothalamic tract, Lateralspinothalamic tract, Anterior spinothalamic tract, or any combinationthereof.

Nucleic acids, vectors, and/or compositions to be used can be utilizedin liquid or freeze-dried form (in combination with one or more suitablepreservatives and/or protective agents to protect the virus during thefreeze-drying process). For gene therapy (e.g., of neurologicaldisorders which may be ameliorated by a specific gene product) atherapeutically effective dose of nucleic acids, vectors, and/orcompositions expressing the therapeutic protein is administered to ahost in need of such treatment. The use of the nucleic acids, vectors,and/or compositions provided herein in the manufacture of a medicamentfor inducing immunity in, or providing gene therapy to, a host is withinthe scope of the present application.

A therapeutically effective amount of the nucleic acids, vectors, and/orcompositions provided herein can be administered to a subject at variouspoints of time. For example, the nucleic acids, vectors, and/orcompositions provided herein can be administered to the subject priorto, during, or after the subject has developed a disease, disorder,and/or infection. The nucleic acids, vectors, and/or compositionsprovided herein can also be administered to the subject prior to,during, or after the occurrence of a disease, disorder, and/orinfection. In some embodiments, the nucleic acids, vectors, and/orcompositions provided herein are administered to the subject duringremission of the disease or disorder. In some embodiments, the nucleicacids, vectors, and/or compositions provided herein are administeredprior to the onset of the disease or disorder in the subject. In someembodiments, nucleic acids, vectors, and/or compositions provided hereinare administered to a subject at a risk of developing the disease ordisorder.

The dosing frequency of the nucleic acids, vectors, and/or compositionsprovided herein can vary. For example, nucleic acids, vectors, and/orcompositions provided herein can be administered to the subject aboutonce every week, about once every two weeks, about once every month,about one every six months, about once every year, about once every twoyears, about once every three years, about once every four years, aboutonce every five years, about once every six years, about once everyseven years, about once every eight years, about once every nine years,about once every ten years, or about once every fifteen years. In someembodiments, the nucleic acids, vectors, and/or compositions providedherein are administered to the subject at most about once every week, atmost about once every two weeks, at most about once every month, at mostabout one every six months, at most about once every year, at most aboutonce every two years, at most about once every three years, at mostabout once every four years, at most about once every five years, atmost about once every six years, at most about once every seven years,at most about once every eight years, at most about once every nineyears, at most about once every ten years, or at most about once everyfifteen years.

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in furtherdetail in the following examples, which are not in any way intended tolimit the scope of the present disclosure.

Example 1 Proof-of-Principle for Protein-Level Incoherent Feed-ForwardLoop Circuits

This example provides experimental demonstration of protein-levelcatalytic incoherent feed forward loops which show dosage compensatingexpression.

Single transcript protein-level IFFLs consisting of a protein with afused N-degron tag, a protease which cleaves the N-degron tag, and anunregulated mCherry to read out the total gene dosage, all co-producedvia 2A peptide sequences (shown in FIG. 1A) were cloned and tested. AnEf1a promoter drives the expression of (1) the protein of interest(payload protein) (“Citrine”), which is fused to an N-end degron(“N-degron”) concealed by a TEVP cleavage site (“ENLYFQY”; SEQ ID NO:5), (2) the co-translated protease (“TEVP”) triggers the degradation ofthe regulated protein (payload protein) (“Citrine”) by cleaving itssubstrate (“ENLYFQY”; SEQ ID NO: 5), and (3) an unregulated fluorescentprotein (“mCherry”) is included to provide a measurement of thetranscriptional dosage. The components were separated by T2A and P2Aself-cleaving peptide sequences. Transcription was terminated with anhGH polyadenylation sequence. FIG. 1B depicts fluorescence microscopyimages showing de-correlated input and output as a result of the IFFL.Boxes emphasize adjacent cells where input (mCherry) is very differentbut output (Citrine) is very similar, compared to controls where bothinput and output vary significantly. FIG. 1C depicts exemplary datashowing that tunability is established using different amino acidsequences for the TEVP cleavage site. The steady state expression of thegene is determined by the k_(cat)/K_(m) of the protease, which can bevaried by changing the sequence of the protease cleavage site. In thisexample, 3 peptide sequences are chosen with different k_(cat)/K_(m) forTEVP. A numerical simulation of the steady state gene expression at eachof these levels is shown in FIG. 1D. FIG. 1E depicts experimentalconfirmation of numerical and analytic models of TEVP performance usingflow cytometry. HEK293 cells were transiently transfected with plasmidDNA containing the IFFL circuits and after a period of 2 days wereanalyzed using a flow cytometer. Data is divided into 20 bins percondition, dots indicate the median of this bin, error bars are ±1standard deviation (in log space) of the bin, the dotted lines are curvefits to the analytical model in Equation (1) shown in Example 5. Flowcytometry measurements on HEK293 cells transiently transfected withthese circuits show expression that agrees strikingly with theanticipated results, and leads to steady state expression that can betuned by adjusting the peptide sequence of the protease cleavage site(FIGS. 1B-1E). The tunability is based on sequence and independent ofviral titer, so it should be robust to many of the challenges of Rettsyndrome gene therapy and designable to express MeCP2 at the correctlevel.

It was found that MeCP2 could not be regulated by the N-terminal tag: anIFFL construct with MeCP2-EGFP being regulated produced proportionalMeCP2-EGFP and mCherry expression (data not shown). It was hypothesizedthat this could be due to inaccessibility of the N-terminus of MeCP2 dueto its chromatin incorporation. To test this, the output of a nuclearlocalized IFFL regulating H2B-Citrine, which is incorporated intochromatin, was compared against a nuclear localized IFFL regulating aCitrine with NLS tags on each terminus (“NLS-Citrine”), which is notincorporated into chromatin. It was found that H2B-Citrine was notregulated by the IFFL while NLS-Citrine construct functioned as in FIG.1 (not shown), supporting the chromatin incorporation hypothesis. Toavoid the chromatin protection, an MeCP2 construct was then designedwith a cut site in the center of the protein, between N-terminus—whichbinds to DNA and chromatin—and a C-terminal co-repressor domain, and itwas found that this construct displayed adaptive expression (FIGS.2A-2B). A H2B-Citrine IFFL (“H2B-Citrine” or “H2B-Cit”) construct wasgenerated comprising of an Ef1a promoter driving the expression of anH2B-Citrine fusion with an N-degron tag on the N-terminus, co-producedwith a TEVP protease with NLS sequences on each terminus as well as anunregulated mCherry, also with NLS sequences on each terminus (FIG. 2A).A Split-MeCP2 IFFL (“Split-MeCP2”) construct was generated comprising anEf1a promoter driving the expression of an mCherry fused to theN-terminus of an MeCP2 gene with a TEVP cleavage site and N-degroninserted into a glycine/serine rich central region between theN-terminal DNA binding domain and the C-terminal NCoR/SMRT co-repressordomain with an EGFP fused past the C-terminus. A TEVP with NLS sequenceson each terminus is co-produced via a self-cleaving T2A peptidesequence. These constructs were transiently transfected into HEK293cells and were analyzed via flow cytometry after a period of 2 days. TheH2B-Cit construct was protected from regulation by the N-degron due tochromatin incorporation, and produced proportional Citrine fluorescenceand mCherry fluorescence. The split MeCP2 construct showed adaptiveexpression of MeCP2-EGFP. Both these methods function in the cytoplasmand nucleus and can, in principle, regulate any proteins localizedthere.

As further proof-of-principle the citrine-regulating circuit waspackaged in two different AAV vectors: AAV9 and PHP.eB, and used them totransduce both HEK293s and a mouse glioblastoma cell line (N2A) at twodifferent titers. The protein-level IFFL from FIG. 1 was packaged intoAAV9 or PHP.eB vectors and transduced into different cell lines—N2A(mouse glioblastoma cells) and HEK293 (human embryonic kidney cells) atdifferent titers (FIG. 3A). Briefly, AAV viruses were produced in HEK293cells, purified, and titered using a protocol developed for producingAAV vectors for use in rodents. These purified vectors were added toHEK293 or N2A cells in 24 well plates at titers of 10⁹ or 10¹⁰ viralgenomes per well. After 2 days of incubation, the cells were analyzedusing flow cytometry. Despite the many differences in conditions, theprotein-level IFFL yields the same input-output (mCherry-YFP) curve inexpression of the gene of interest (here, YFP). Despite the variety ofcapsid type, species of cell, and titer of vector, it was found that thegene expression followed the same input-output curve in each case (FIG.3B). Thus, the protein-level IFFL provided herein can function as ageneral module for tunable protein-expression that is robust andreliable across widely different conditions.

Example 2 Proof-of-Principle for miRNA-Level Incoherent Feed-ForwardLoop Circuits

This example provides experimental demonstration of miRNA-levelcatalytic incoherent feedforward loops which show dosage compensatedexpression.

For gene therapy purposes, it can be advantageous to use regulatorymethods that do not involve the production of any foreign proteins,which could be immunogenic. This Example describes the testing miRNAIFFLs that achieve dosage compensation without the production ofproteins besides the gene of interest by incorporating miRNA targetsites as well as a synthetic intron containing an miRNA hairpin sequenceinto the 3′ UTR. The steady state expression of these circuits was tunedby varying the complementarity and copy number of miRNA target sites inthe 3′ UTR. These miRNA IFFL cassettes were used to create circuits with3 levels of regulated expression of both GFP (FIG. 4A) and an MeCP2-EGFPfusion (FIGS. 5A-5B). FIG. 4A shows different constructs were designedto provide 4 different GFP expression behavior. All expressionconstructs are based on an original construct (“Control”) whichcomprises an Ef1a promoter driving mCherry, terminated by a bGHpolyadenylation sequence, followed by an insulator, followed by a CMVpromoter driving the expression of GFP, terminated by an hGHpolyadenylation sequence. First from top of FIG. 4A: Control circuit(“Control”) where GFP expression is proportional to mCherry. Second fromtop of FIG. 4A (“miR8”): an miRNA expression cassette (miRL) is insertedinto the 3′ UTR as well as a weak target with 8 bp of complementarity.Third from top of FIG. 4A (“miR22”): the 8 bp target is switched out fora 22 bp target, which results in significantly stronger regulation.Fourth from top of FIG. 4A (“miR22-10×”): the 22 bp target is repeated10 times to yield the strongest regulation and the lowest GFPexpression. Thus, tunability is established by varying thecomplementarity and copy number of the miRNA target sites. FIG. 4Bdepicts flow cytometry data showing the 4 different levels ofexpression. HEK293 cells were transiently transfected with theseconstructs and analyzed on a flow cytometer after a period of 2 days.FIGS. 5A-5B show tunable control of MeCP2 Expression using a syntheticmiRNA IFFL. FIG. 5A shows the same constructs from FIG. 4A with anMeCP2-EGFP fusion swapped in for the GFP. FIG. 5B depicts data relatedto HEK293 cells transiently transfected with these constructs andanalyzed on a flow cytometer after a period of 2 days. Flow cytometrydata shows 4 levels of MeCP2-EGFP expression. These miRNA IFFLs can thusbe used to express arbitrary gene cassettes at defined levels, includingMeCP2 as well as large multi-protein circuits, in gene and celltherapies.

Example 3 IFFL Circuit Motif Modulates Expression of Virally DeliveredCargo in Mammals

This example provides in vivo proof-of-principle experimentaldemonstration of miRNA-level catalytic incoherent feedforward loopswhich show dosage compensated expression. FIGS. 6A-6B depictnon-limiting exemplary embodiments and data related to an IFFL circuitmotif modulating the expression of virally delivered cargo in mammals.AAV genomes containing GFP under IFFL control with either 0 or 10 miRNAtarget sites were packaged into AAV-CAP.B22 (Flytzanis et al 2020).AAV-IFFL was then delivered via direct injection (1 uL of 5e12 vg/mL)into the cortices of 8 week old male C57Bl6J mice (FIG. 6A). After 14days of expression, animals were perfused with 4% PFA and brain tissuewas collected in 50 uM slices for imaging. Virally transduced cells areindicated by mRuby expression (FIG. 6B; column 1). At similar levels ofRFP, the genomes containing 10 miRNA target sites (FIG. 6B; row 2)exhibit marked repression of EGFP, consistent with observations ofcircuit behavior in vitro. This experiment shows that shows the 10×IFFLcircuit successfully regulates the expression of Citrine in a livingmouse brain. These mice were behaviorally normal, indicating that theIFFL cassette did not have an adverse effect on mice health.

Example 4 miRNA-Level and Protein-Level Incoherent Feed-Forward LoopCircuits

This example provides in vivo proof-of-principle experimentaldemonstration of additional miRNA-level and protein-level incoherentfeedforward loops described herein which show dosage compensatedexpression.

There different protein-level circuits (FIGS. 7A, 8A, and 9A) wereconstructed comprising different promoters. FIG. 7A depicts aprotein-level IFFL driven by the CMV promoter. The canonical TEVP cutsite, ENLYFQY (SEQ ID NO: 5), was modified to give different steadystate levels in the stable regime, as seen in FIG. 7B. FIG. 8A depicts aprotein-level IFFL driven by the full Ef1a promoter (with intron). Thisconstruct yielded high expression, and similar functional form to thesame construct driven by the CMV promoter (FIG. 8B). In this experiment,the terminal residue in the TEVP cut site was varied, and a veryeffective terminal residue was discovered: ENLYFQF (SEQ ID NO: 6). FIG.9A depicts a protein-level IFFL driven by Ef1a promoter (without theintron). This promoter expressed at a much lower level than the CMV orfull Ef1a promoters, but yielded a similar steady state for thecanonical cut site (FIG. 9B). This construct was the construct used inthe AAV IFFL experiments described above, to yield similar steady stateson different species of cells, titers of virus, and capsids. In sum,despite some variations in the overall expression of the constructs, theoutput expression of the gene of interest (Citrine) followed the samefunctional form.

Three different miRNA-level circuits (FIGS. 10A, 11A and 12A) wereconstructed comprising different promoters. FIG. 10A depicts a firstembodiment of a miRNA-level IFFL circuit wherein expression is driven bytwo separate promoters: Ef1a, which drives the unregulated mCherryexpression, and the CMV promoter, which drives the regulated MeCP2-EGFPexpression. As seen in FIG. 10B, varying the number of miRNA targetsites modulates payload expression. FIG. 11A depicts a second embodimentof a miRNA-level IFFL circuit, optimized to fit inside of an AAV genome.To fit, one bidirectional promoter was constructed by fusing twopromoters back to back: Ef1a (without intron) and MeP229, a minimalMeCP2 promoter, which was chosen because it is small. FIG. 11B depictsdata related to expression from these construct with a variable numberof target sites. FIG. 12A depicts a third embodiment of a miRNA-levelIFFL circuit, wherein the construct has the CMV enhancer insertedbetween the previous two promoters. As seen in FIG. 12B, this circuitcomposition greatly raised the overall expression of the construct ascompared to the construct tested in FIGS. 11A-11B. However, thefunctional form of steady state levels of the previous construct weremaintained.

FIG. 13 illustrates a non-limiting exemplary illustration of amiRNA-level IFFL circuit engineered to reduce endogenous MeCP2expression. By designing the miRNA sequence to target the endogenousMeCP2 transcript, it can be engineered so that the endogenous expressionis knocked out and only the regulated expression remains.

Example 5 Exemplary Modeling Robust Gene Expression

An analytic model was derived for the steady state behavior of aprotein-level catalytic incoherent feed-forward loop showing dosagecompensating behavior. Using ordinary differential equations, thebehavior of a circuit was modeled where a protein of interest is cleavedby a co-produced protease, revealing an N-degron which targets theprotein for proteasomal degradation. In this Example, we simulate thesedifferential equations and solve for the steady state, both of whichshow adaptation to the gene dosage. The steady state expression of theprotein of interest is given by the expression below, which is dependenton the protease Michaelis Menten parameters K_(m) and k_(cat), itsdegradation rate γ, the faster proteasomal degradation rate γ_(deg), andD, the unregulated steady state expression at that gene dosage:

$\begin{matrix}{P_{ss} = {{\frac{K_{m}\gamma}{k_{cat}}\frac{D}{{K_{m}{\gamma/k_{cat}}} + D}} + \frac{D\gamma}{\gamma_{\deg}}}} & (1)\end{matrix}$

which implies that when D gets much larger than K_(m)γ/k_(cat), but lessthan K_(m) γ_(deg)/k_(cat), the expression of the protein of interestsaturates at P_(ss)=K_(m)γ/k_(cat), independent of D. Thus, in thatregime, the circuit adapts to different gene dosages, expressing theprotease at the same level.

INTRODUCTION

In gene therapy it is difficult to control the level of payload thatgets delivered to any individual cell. In several applications,deleterious effects are associated with over-dosing the delivered gene.For example, gene therapy for Rett syndrome would deliver a functioningversion of MECP2 to neurons of an afflicted patient. However,duplication of the MECP2 gene causes the harmful MECP2 duplicationsyndrome, which would suggest that neurons are sensitive to even 2-foldexcesses of MECP2. Another complication is that, since Rett syndrome isan X-linked heterozygous disease, 50% of neurons are expressing healthylevels of MECP2. Therefore, any gene therapy for Rett syndrome mustcarefully regulate the amount of delivered gene that is expressed toensure that total levels are in the “Goldilocks” zone. However, it hasyet been unclear what kind of regulation would be sufficient to yield anexpression profile that saturates past some level of dosage.

In this Example a simple system is modeled where MECP2-degron-tevs iscoexpressed with a TEVP, and it is shown analytically and throughsimulation that this system is sufficient to yield expression of MECP2that saturates at a high dosage level. Without being bound by anyspecific theory, it is believed that:

1. for MECP2 production to overwhelm the TEVP, in some embodiments, atleast 10⁴ molecules of MECP2 are required to be produced for each TEVPmolecule for realistic parameters (each TEVP can cleave around 10⁴substrates by the time it degrades).

2. at large levels of dosage, the concentration of uncleaved MECP2approaches and stays beneath an asymptote, and this asymptote is small(6.2×10⁻⁴ WT MECP2 concentration for realistic parameters). In someembodiments, this asymptote can be pushed higher by reducing thek_(cat)/K_(M) of the protease—it would need to be reduced 1000-fold toget the desired asymptote.

3. the level of cleaved MECP2 scales with the dosage, but thispopulation is insignificant.

A Simple ODE Model

The dynamics of the system are assumed to be standardproduction-degradation with Michaelis-Menton kinetics describing thecleavage of uncleaved MECP2 (M) by TEVP (T) to produce cleaved MECP2(M_(deg)). The dosage level of the system is parameterized by theproduction rate D. A scalar parameter α modifies the production rate ofMECP2 to account for a situation where multiple MECP2s are produced foreach TEVP (MECP2-P2A-MECP2-T2A-TEVP). All proteins have their respectivedegradation rates γ_(T), γ_(M), γ_(degron). The Michaelis-Mentonparameters for TEVP are k_(cat) and K_(M).

$\overset{.}{T} = {D - {\gamma_{T}T}}$$\overset{.}{M} = {D - {\gamma_{M}M} - {k_{cat}T\frac{M}{K_{M} + M}}}$${\overset{.}{M}}_{deg} = {{k_{cat}T\frac{M}{K_{M} + M}} - {\gamma_{deg}M_{deg}}}$$\overset{.}{T} = {D - {\gamma T}}$$\overset{.}{M} = {D - {\gamma M} - {k_{cat}T\frac{M}{K_{M} + M}}}$${\overset{.}{M}}_{deg} = {{k_{cat}T\frac{M}{K_{M} + M}} - {\gamma_{deg}M_{deg}}}$

To make the equations dimensionless, the time scale was chosen to be1γ_(T). The concentration scale is arbitrary and it was chosen to beM_(WT), the wild-type steady state MECP2 concentration. Definingunitless variables:

${\gamma = {\gamma_{M}/\gamma_{T}}},{\gamma_{deg} = {\gamma_{degron}/\gamma_{T}}},{k = {k_{cat}/\gamma_{T}}},{K = \frac{K_{M}}{M_{WT}}},{D = {D/\left( {M_{WT}\gamma_{T}} \right)}},$

the ODE can be made dimensionless:

$\overset{.}{T} = {D - T}$$\overset{.}{M} = {{\alpha\; D} - {\gamma\; M} - {{kT}\frac{M}{K + M}}}$${\overset{.}{M}}_{deg} = {{{kT}\frac{M}{K + M}} - {\gamma_{deg}M_{deg}}}$

The Steady State can be Simplified by Considering Limiting Cases

Since T and M do not depend on M_(deg), it is ignored for now and willbe considered later. For the steady states T_(ss) and M_(ss) thederivatives were set to zero to get:

T_(ss) = D${M_{ss}^{2} + {\left\lbrack {K + {\frac{1}{\gamma}\left( {k - \alpha} \right)D}} \right\rbrack M_{ss}} - \frac{K\alpha D}{\gamma}} = 0$

The quadratic formula can be used to solve for M_(ss) (the minus roothas been eliminated by proving that it is always negative, proofomitted):

$M_{ss} = {\frac{1}{2}\left\lbrack {{- \left\lbrack {K + {\frac{1}{\gamma}\left( {k - \alpha} \right)D}} \right\rbrack} + \sqrt{\left( {K + {\frac{1}{\gamma}\left( {k - \alpha} \right)D}} \right)^{2} + {4\frac{K\alpha D}{\gamma}}}} \right.}$

The complicated form of this solution is an obstacle to analysis.However, we are mostly concerned with limiting cases when D gets verylarge. In those situations, the 1/γ(k−α)D terms dominate, and thebehavior depends on the sign of (k−α).

Case of interest 1: k−α<0, D>>K, MECP2 expression runs away with dosage

If k<α, more MECP2 is created per unit of TEVP than can be catalyticallydegraded by TEVP by the time that it degrades. This case captures theworry that there is some level of MECP2 expression that cannot becontained by TEVP.

In the regime where D is large, the

$\frac{1}{\gamma}\left( {k - \alpha} \right)$D term dominates the other terms in the square root, leading to theapproximation:

$M_{ss} \approx {{- {\frac{1}{2}\left\lbrack {\frac{1}{\gamma}\left( {k - \alpha} \right)D} \right\rbrack}} + {\frac{1}{2}{{{\frac{1}{\gamma}\left( {k - \alpha} \right)D}}.}}}$

This can be further simplified to:

$M_{ss} \approx {\frac{\left( {\alpha - k} \right)}{\gamma}D}$

Thus the expression of MECP2 is linearly dependent on the dose in thisregime. However, this regime is hard to get to. Since α≈1 for ourdesigns, this regime would mean that k_(cat) for TEVP is on the order ofits degradation rate or slower. This is orders of magnitude slower thanit really is. Thus we believe we can be comfortable assuming we are inthe situation where k−α>0.

Case of Interest 2: k−α>0, D>>K, MECP2 Expression Approaches Asymptote

When k−α>0, we cannot use the same trick as before since the answercomes out to 0—we must consider the higher order terms. We factor thesquared term out of the square root and this gives me a term with D inthe denominator, so it must be very small. We use small argumentexpansion on the square root (√{square root over (1+x)} ≈1+x/2 for smallx) to get:

$\begin{matrix}{M_{ss} = {\frac{1}{2}\left\lbrack {{- \left\lbrack {K + {\frac{1}{\gamma}\left( {k - \alpha} \right)D}} \right\rbrack} +} \right.}} \\\left. {\left( {K + {\frac{1}{\gamma}\left( {k - \alpha} \right)D}} \right)\sqrt{1 + \frac{4K\alpha}{D\;\gamma\;\left( {\frac{K}{D} + {\frac{1}{\gamma}\left( {k - \alpha} \right)}} \right)^{2}}}} \right\rbrack \\{\approx {\frac{1}{2}\left\lbrack {{- \left\lbrack {K + {\frac{1}{\gamma}\left( {k - \alpha} \right)D}} \right\rbrack} +} \right.}} \\\left. {\left( {K + {\frac{1}{\gamma}\left( {k - \alpha} \right)D}} \right)\left( {1 + \frac{2K\;\alpha}{D\;\gamma\;\left( {\frac{K}{D} + {\frac{1}{\gamma}\left( {k - \alpha} \right)}} \right)^{2}}} \right)} \right\rbrack\end{matrix}$$M_{ss} \approx {\frac{K\;\alpha}{k - \alpha}\frac{D}{{\gamma\;{K/\left( {k - \alpha} \right)}} + D}}$

The asymptotic limit as D→∞ is:

$M_{asym} = {{\frac{K\alpha}{k - \alpha} \approx {\frac{K}{k}\mspace{14mu}{for}\mspace{14mu}\alpha}} = 1}$

Thus we have a system that approaches but never exceeds the ratio of Kand k, a good rule of thumb.

${M_{ss} \approx {\frac{K_{M}}{{k_{cat}/\gamma} - 1}\frac{D}{\frac{\gamma\; K_{M}}{{k_{cat}/\gamma} - 1} + D}}}\overset{\;{D\rightarrow\infty}\mspace{11mu}}{\rightarrow}\frac{\gamma K_{M}}{k_{cat}}$$D_{\max} = {\frac{K_{M}}{{k_{cat}/\gamma_{T}} - 1}\left( {\gamma_{\deg} - \gamma_{M}} \right)}$$D_{thresh} = \frac{\gamma_{M}K_{M}}{{k_{cat}/\gamma_{T}} - 1}$${Range} = \frac{\gamma_{\deg} - \gamma_{M}}{\gamma_{M}}$${M_{ss} \approx {\frac{\gamma K_{M}}{k_{cat}}\frac{D}{\frac{\gamma\; K_{M}}{k_{cat}} + D}}}\overset{\;{D\rightarrow\infty}\mspace{11mu}}{\rightarrow}\frac{\gamma K_{M}}{k_{cat}}$$\frac{D_{\max}}{D_{thresh}} = \frac{\gamma_{\deg}}{\gamma_{M}}$

Cleaved Population Scales with D

Returning to the cleaved MECP2 component of the ODE:

${\overset{.}{M}}_{deg} = {{{kT}\frac{M}{K + M}} - {\gamma_{deg}M_{deg}}}$

We can solve for the steady state in terms of the asymptotic steadystate of M:

$\begin{matrix}{M_{deg} = {\frac{kD}{\gamma_{deg}}\frac{M_{ss}}{K + M_{ss}}}} \\{= {\frac{kD}{\gamma_{deg}}\frac{K{\alpha/\left( {k - \alpha} \right)}}{K + {K{\alpha/\left( {k - \alpha} \right)}}}}}\end{matrix}$ $M_{deg} \approx \frac{D}{\gamma_{deg}}$

Thus the population of cleaved MECP2 scales with the dosage, but has areasonably large divisor in γ_(deg). This technically breaks the“dosage-independence” but if the degradation rate is significantly fastthen the slope will be very small.

Choosing Realistic Parameters

MECP2 Concentration Scale

From Skene et al. (Molecular cell, 2010) describes that “The amount ofMeCP2 in sorted neuronal nuclei (NeuN positive) was 16×10⁶ molecules pernucleus”, which is 2.7×10⁻¹⁷ mols. Modeling the nucleus as a sphere of10 micrometers in diameter, the volume is:¾π(10×10⁻⁶ m)³=4.19×10⁻¹⁵ m³=4.19×10⁻¹² liters.

To compute the molarity we divide the mols by the volume in liters toget:

$M_{WT} = {\frac{{2.7} \times 10^{{- 1}7}\mspace{14mu}{mols}}{{4.1}9 \times 10^{{- 1}2}\mspace{14mu}{liters}} = {6.4\mspace{14mu}{{\mu M}.}}}$

TEVP Kinetics

Kapust et al. (Protein engineering, 2001) list the TEVP K_(M) valuesranging around 50 μM and k_(cat) values ranging around 0.15 s⁻¹.

Degradation Rates

Bionumbers lists the average protein half life in human cells as around30 hours. Yen, Hsueh-Chi Sherry, et al. (Science, 2008) lists the halflife of a GFP with a degron as 1 hour. Based on these numbers we set:γ_(T)=γ_(M)=(1/30) hr⁻¹ and γ_(degron)=1 hr⁻¹.

Final Parameter List:

-   -   α≈1 There is one MECP2-T2A-TEVP    -   γ≈1 this Example assumes there is no significant difference        between the degradation rate of TEVP and MECP2    -   γ_(deg)≈100 degron increases degradation rate approximately        100-fold.    -   K=10 because the measured TEVP K_(m) value is about 10 times the        WT neural concentration of MECP2.    -   k=0.15 s⁻¹/(1/30) hr⁻¹=1.62×10⁴ One TEVP molecule can cleave        around 10⁴ substrates by the time it degrades.

Simulation

Given the parameters above, the circuit was simulated with the codeshown in FIG. 14A for dosages D equal to 1, 10, 30, and 100. Theuncleaved population, after a sharp peak, settles to an asymptote thatseems to agree with the formula M_(asym)=K/k=0.00062. The cleavedpopulation dominates with 10⁴ times higher concentration and seems toagree with the formula M_(deg)=D/γ_(deg)=D/30, and thus up to D=30, itseem to be within reasonable dosage range for these parameters. FIG.14A-14C depict non-limiting exemplary code and simulations.

Checking the Functional Form of the Dosage-Expression Relationship

Next, it was checked how well the approximation

$P_{ss} = {{\frac{K_{M}\gamma}{k_{cat}}\frac{D}{{\gamma\frac{K_{M}\gamma}{k_{cat}}} + D}} + \frac{D}{\gamma_{\deg}}}$

fits the output of the simulation. FIGS. 15A-15B, 16A-16B, and 17A-17Bdepict non-limiting exemplary code and simulations.

Confirming that the Uncleaved Asymptote Scales with K/k:

As seen in FIGS. 18A-18B and 19A-19B, which depict non-limitingexemplary code and simulations, we varied K or k and checked the formulaM_(asym)=K/k by labeling the location with a blue dashed line. In allcases the formula works well.

Confirming that Runaway Threshold is k−α=0

FIGS. 20A-20C depict non-limiting exemplary code and simulations. Thefirst example here is where k−α<0, the second where k−α>0. The firstshould have runaway behavior with dosage, the second, asymptotic. Thisbehavior was confirmed with the plots.

Ideal Situation K/k=0.33

FIGS. 21A-21B depict non-limiting exemplary code and simulations. Thisplot is of a situation where k/K of TEVP has been reduced by around1000-fold and TEVP is purely catalytic. In this case, we get a steadystate concentration of MECP2 that is suitable for gene therapy.

Predicted Behavior for Known Substrates

From Tözseŕ, József, et al. (FEBS, 2005), TEVP k_(cat)/K_(M) values areknown:

ETVFFQ (SEQ ID NO: 1) ≈ 0.007 ENAYFQ (SEQ ID NO: 2) ≈ 0.027ENLFFQ (SEQ ID NO: 3) ≈ 0.35 ENLYFQ (SEQ ID NO: 4) ≈ 4.51

Predicted output concentrations are shown in the non-limiting exemplarycode and simulations of FIGS. 22A-22B.

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. As used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise. Any reference to “or” herein isintended to encompass “and/or” unless otherwise stated.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. A nucleic acid comprising: a promoter operablylinked to a polynucleotide encoding a fusion protein comprising apayload protein, a protease, and one or more self-cleaving peptidesequences, wherein the payload protein comprises a degron and a cut sitethe protease is capable of cutting to expose the degron, wherein thedegron of the payload protein being exposed changes the payload proteinto a payload protein destabilized state, wherein the self-cleavingpeptide sequence comprises porcine teschovirus-1 2A peptide (P2A),Thosea asigna virus 2A peptide (T2A), equine rhinitis A virus 2A peptide(E2A), foot-and-mouth disease virus 2A peptide (F2A), or any combinationthereof, and wherein the protease comprises tobacco etch virus (TEV)protease, tobacco vein mottling virus (TVMV) protease, hepatitis C virusprotease (HCVP), derivatives thereof, or any combination thereof.
 2. Thenucleic acid of claim 1, wherein the degron comprises an N-degron. 3.The nucleic acid of claim 1, wherein one or more cells comprise anendogenous version of a gene encoding the payload protein comprising oneor more secondary silencer binding sequences in the 3′UTR, wherein thenucleic acid comprises a secondary silencer effector cassette encoding asecondary silencer effector that is capable of binding the one or moresecondary silencer binding sequences.
 4. The nucleic acid of claim 1,wherein the polynucleotide further comprises a transcript stabilizationelement, and wherein the transcript stabilization element compriseswoodchuck hepatitis post-translational regulatory element (WPRE), bovinegrowth hormone polyadenylation (bGH-polyA) signal sequence, human growthhormone polyadenylation (hGH-polyA) signal sequence, or any combinationthereof.
 5. The nucleic acid of claim 1, wherein the promoter comprisesa ubiquitous promoter, and wherein the ubiquitous promoter is selectedfrom the group comprising a cytomegalovirus (CMV) immediate earlypromoter, a CMV promoter, a viral simian virus 40 (SV40) (e.g., early orlate), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Roussarcoma virus (RSV) LTR, an RSV promoter, a herpes simplex virus (HSV)(thymidine kinase) promoter, H5, P7.5, and P11 promoters from vacciniavirus, an elongation factor 1-alpha (EF1a) promoter, early growthresponse 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiationfactor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shockprotein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa(HSP70), β-kinesin (β-KIN), the human ROSA 26 locus, a Ubiquitin Cpromoter (UBC), a phosphoglycerate kinase-1 (PGK) promoter,3-phosphoglycerate kinase promoter, a cytomegalovirus enhancer, humanβ-actin (HBA) promoter, chicken β-actin (CBA) promoter, a CAG promoter,a CBH promoter, or any combination thereof.
 6. The nucleic acid of claim1, wherein the promoter is an inducible promoter, wherein the induciblepromoter is a tetracycline responsive promoter, a TRE promoter, a Tre3Gpromoter, an ecdysone responsive promoter, a cumate responsive promoter,a glucocorticoid responsive promoter, and estrogen responsive promoter,a PPAR-γ promoter, an RU-486 responsive promoter, or any combinationthereof.
 7. The nucleic acid of claim 1, wherein the promoter comprisesa tissue-specific promoter, wherein the tissue specific promoter is aliver-specific thyroxin binding globulin (TBG) promoter, an insulinpromoter, a glucagon promoter, a somatostatin promoter, a pancreaticpolypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatinekinase (MCK) promoter, a mammalian desmin (DES) promoter, a α-myosinheavy chain (a-MHC) promoter, a cardiac Troponin T (cTnT) promoter, orany combination thereof.
 8. The nucleic acid of claim 1, wherein thepromoter comprises a tissue-specific promoter, wherein the tissuespecific promoter is a neuron-specific promoter, wherein theneuron-specific promoter comprises a synapsin-1 (Syn) promoter, aCaMKIIa promoter, a calcium/calmodulin-dependent protein kinase II apromoter, a tubulin alpha I promoter, a neuron-specific enolasepromoter, a platelet-derived growth factor beta chain promoter, TRPV1promoter, a Na_(v)1.7 promoter, a Na_(v)1.8 promoter, a Na_(v)1.9promoter, an Advillin promoter, or any combination thereof.
 9. Thenucleic acid of claim 1, wherein the promoter comprises an intronicsequence, a bidirectional promoter and/or an enhancer.
 10. The nucleicacid of claim 1, wherein one or more cells comprise an endogenousversion of the payload gene, and wherein the promoter comprises or isderived from the promoter of the endogenous version.
 11. The nucleicacid of claim 1, wherein the payload protein comprises adisease-associated protein, wherein aberrant expression of thedisease-associated protein correlates with the occurrence and/orprogression of the disease.
 12. The nucleic acid of claim 1, wherein thepayload protein comprises methyl CpG binding protein 2 (MeCP2), DRK1A,KAT6A, NIPBL, HDAC4, UBE3A, EHMT1, one or more genes encoded onchromosome 9q34.3, NPHP1, LIMK1 one or more genes encoded on chromosome7q11.23, P53, TPI1, FGFR1 and related genes, RAL SHANK3, CLN3, NF-1,TP53, PFK, CD40L, CYP19A1, PGRN, CHRNA7, PMP22, CD40LG, derivativesthereof, or any combination thereof.
 13. The nucleic acid of claim 1,wherein the payload protein comprises a programmable nuclease, adiagnostic agent, a chimeric antigen receptor, a CRE recombinase, GCaMP,a cell therapy component, a knock-down gene therapy component, acell-surface exposed epitope, or any combination thereof.
 14. Acomposition comprising the nucleic acid of claim 1, wherein thecomposition is a vector, a ribonucleoprotein (RNP) complex, a liposome,a nanoparticle, an exosome, a microvesicle, or any combination thereof.15. The composition of claim 14, wherein the vector is a viral vector, aplasmid, a naked DNA vector, a lipid nanoparticle, or any combinationthereof.
 16. A method of treating a disease or disorder in a subject,the method comprising: introducing into one or more cells of a subjectin need thereof the composition of claim
 14. 17. The method of claim 16,wherein, in the absence of the protease and/or the silencer effector,the payload protein reaches untuned steady state payload protein levelsin the one or more cells, wherein untuned steady state payload proteinlevels range between a lower untuned threshold and an upper untunedthreshold of an untuned expression range, wherein, in the presence theprotease and/or the silencer effector, the payload protein reaches tunedsteady state payload protein levels in the one or more cells, whereintuned steady state payload protein levels range between a lower tunedthreshold and an upper tuned threshold of a tuned expression range,wherein the difference between the lower untuned threshold and the upperuntuned threshold of the untuned expression range is greater than abouttwo orders of magnitude, and wherein the difference between the lowertuned threshold and the upper tuned threshold of the tuned expressionrange is less than about one order of magnitude.
 18. The method of claim17, wherein the lower tuned threshold and/or the upper tuned thresholdof a tuned expression range can be increased by introducing one or morenon-canonical amino acid substitutions into the cut site, and whereinthe lower tuned threshold and/or the upper tuned threshold of a tunedexpression range can be reduced by introducing one or more canonicalamino acid substitutions into the cut site.